Monensin methods

Marecek and colleagues developed a new electrochemical method for the rapid quantitative analysis of the antibiotic monensin in the fermentation vats used during its production. The standard method for the analysis, which is based on a test for microbiological activity, is both difficult and time-consuming. As part of the study, samples taken at different times from a fermentation production vat were analyzed for the concentration of monensin using both the electrochemical and microbiological procedures. The results, in parts per thousand (ppt), are reported in the following table. 

This is an example of a paired data set since the acquisition of samples over an extended period introduces a substantial time-dependent change in the concentration of monensin. The comparison of the two methods must be done with the paired f-test, using the following null and two-tailed alternative hypotheses... 

Because of the complexity of the polyether antibiotics tittle progress has been made in stmcture determination by the chemical degradation route. X-ray methods were the techniques most successfully applied for the early stmcture elucidations. Monensin, X206, lasalocid, lysocellin, and salinomycin were included in nineteen distinct polyether x-ray analyses reported in 1983 (190). Use of mass spectrometry (191), and H (192) and nmr (141) are also reviewed. More recently, innovative developments in these latter techniques have resulted in increased applications for stmcture determinations. Eor example, heteronuclear multiple bond connectivity (hmbc) and homonuclear Hartmann-Hahn spectroscopy were used to solve the stmcture of portimicin (14) (193). East atom bombardment mass spectrometry was used in solving the stmctures of maduramicin alpha and co-factors (58). 

Some of these compounds could be considered as dietary additives, but various other terms, including pesticides, can also be used. They can have beneficial effects on the environment and this aspect will be discussed later. The ionophore monensin, which is an alicyclic polyether (Figure 1), is a secondary metabolite of Streptomyces and aids the prevention of coccidiosis in poultry. Monensin is used as a growth promoter in cattle and also to decrease methane production, but it is toxic to equine animals. " Its ability to act as an ionophore is dependent on its cyclic chelating effect on metal ions. ° The hormones bovine somatotropin (BST) and porcine somatotropin (PST), both of which are polypeptides, occur naturally in lactating cattle and pigs, respectively, but can also be produced synthetically using recombinant DNA methods and administered to such animals in order to increase milk yields and lean meat production. "... 

The coccidiostat monensin (Table 7.1, XXVII) dissolved in nitrobenzene with a dilute HNOj + NaCl solution as aqueous phase electrolyte can be accurately determined by this method, also in Streptomyces culture extracts [ 1 la]. 

Weiss and MacDonald (87) recently reviewed methods for determination of ionophore antibiotics. lonophores approved for use in animal agriculture in the U.S. are lasalocid, monensin, and salinomycin. An HPLC ( ) and GLC-MS ( ) procedure have been described for lasalocid. For other ionophores, TLC-bioautography is the preferred procedure because of lack of any useful UV absorbance. However, a few TLC colorimetric procedures have been described for monensin residues in tissues (90-92). 

Complex formation constants measured by several groups using various methods are summarized in Table 2 (for full details see (56)). The antibiotics tabulated prefer K+ relative to Na+, except monensin and antamanide, which prefer Na+ relative to K+. These findings are in agreement with ion-selectivity sequences observed in biological systems (6, 57-61). 

Examples exist of different types of immunobased technology being interfaced to produce an effective analytical system. In a variety of recent methods, immunoaffinity chromatography has been employed for purification, chemiluminescence enzyme immunoassay has been used for quantification of salbutamol and clenbuterol in tissue and plasma from calves and pigs (122), clenbuterol in cattle hair (123), and monensin in chicken tissues (124). In these methods, quantification at sub-ppb levels has been demonstrated. 

The cleavage of acyclic acetals by N-bromosuccinimide was discovered in 1951.128 The reaction was subsequently extended to cyclic derivatives129 but its synthetic potential was not fully realised until Hanessian applied the method to carbohydrate benzylidene acetals to give bromo benzoate esters.130 A noteworthy feature of the transformation is that it can be performed on a 100 g (or larger) scale [Scheme 374],131-132 The reaction has been exploited in the synthesis of a number of natural products such as Monensin,133 Pseudomonic acid,134 Boromycin,135 Thienamycin.136 and Rifamycin.137 Williams and co-workers42... 

Bromoetherification, the addition of the elements of Br and OR to a double bond, is a common method for constructing rings containing oxygen atoms. This reaction has been used in the synthesis of the polyether antibiotic monensin (Problem 21.40). Draw a stepwise mechanism for the following intramolecular bromoetherification reaction. 

The step boxed in yellow in the ester reduction scheme on p. 000 gave an aldehyde. The aldehyde is more readily reduced than the ester, so the reduction doesn t stop there, but carries on to the alcohol oxidation level. How, then, can you reduce an ester to an aldehyde This is a real problem in synthetic chemistry—the ester below, for example, is easy to make by methods you will meet in Chapter 27. But an important synthesis of the antibiotic monensin requires the aldehyde. 

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for maduramicin in poultry feed. The assay utilized polyclonal anti-maduramicin antibody raised in rabbits, maduramicin monoamide with 1,6-hexane diamine-conjugated ovalbumin as the coating antigen, horseradish peroxidase conjugated goat anti-rabbit IgG and 2,2 azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) for quantitation. Standard curves ranging from 0 to 80 ng/mL maduramicin were constructed. The assay did not cross-react with monensin, lasalocid, salinomycin, lincomycin, narasin, chlortetracycline or roxarsone. Broiler feed fortified at 4 to 7 ppm maduramicin were shown to be quantifiable by ELISA at an average recovery of 98.1%. This ELISA method for maduramicin in poultry feed is comparable to the established HPLC-F method. 

From Eq. (31.2), we can evaluate the pH of Hb. salinarum without ratio methods. The fluorescence lifetime in Hb. salinarum without monensin is evaluated to be 2.76 ns. The intracellular pH is then calculated to be 7.1, which is in reasonable agreement with that obtained from the excitation ratio method [18]. The reason why the fluorescence lifetime in vivo is smaller than that in vitro may be ascribed to the local field produced by some proteins and membranes that affects the... 

In this study we have used monensin primarily as a means to perturb this ecosystem in order to evaluate our methods for measuring... 

Monensin was the first polyether antibiotic to have its structure solved by crystallographic methods. In the silver salt, the monensin anion is wrapped around the... 

Other veterinary drugs Other veterinary drugs of importance are the anticoccidial feed additives such as the ionophores, narasin, monensin, salinomycin, apramycin, lasalocid, and nicarbazin. The USDA/ FSIS have a method for the major ionophores in tissue samples, which is based on purification of sample extracts by silica gel, alumina, or ion-ex-change column chromatography and determination by TEC with detection by bioautography. A number of alternative methods based on immunoassays, biosensor technology, and HPLC have been developed. 

In the case of ionophore determination a rather high concentration of the ion to be complexed in the aqueous phase is used while the ionophore is present at a low concentration in the organic phase. Under increasing potential scan the current peak is controlled by diffusion of the ionphore to ITIES and by diffusion of the complex formed from ITIES into the bulk of the organic phase while after scan reversal opposite processes take place. The peak currents are proportional to ionophore concentration. This method has been applied to the determination of monensin in cultures of Streptomyces cinnamonensis [33]. 

---