Penicillin assay

Place six cylinders on the inoculated agar surface so that they are at approximately 60° intervals on a 2.8 cm. radius. Use three plates for each sample. FiU three cylinders on each plate with the 20 /ig. r milliliter standard and three cylinders with the 20 /ig. per milliliter (estimated) sample, alternating standard and sample. At the same time prepare a standard curve using concentrations of the standard of 36.0, 32.0, 28.0, 24.0, 20.0,16.0,12.0,10.0, and 8.0 />g. per milliliter in 1% phosphate buffer, pH 6.0. A total of 24 plates is used in the preparation of this standard curve, three plates for each solution, except the 20 /ig. per milliliter solution. The latter concentration is used as the reference point and is included on each plate. On each of three plates fill three cylinders with the 20 /ig. per milliliter standard and the other three cylinders with the concentration of the standard under test. Thus, there will be seventy-two 20 Mg. determinations and nine determinan tions for each of the other points on the curve. Incubate the plates for 16-18 hours at 32-35 °C. and measure the diameter of each zone of inhibition. Correct the average value for each point in the same manner as described for the penicillin assay (section 11,1). 

Culture and Inoculiun. Stock cultures of Sardna IvJtea (P. C. I. 1001) are carried on agar slants having the isame composition as the stock culture medium described for penicillin assay (Section 11,1), and the inoculum suspension may also be prepared in the broth described in that same section. 

Place six cylinders on the inoculated agar surface so that they are at approximately 60° intervals on a 2.8-cm. radius. Use three plates for each sample. Fill three cylinders on each plate with the 100 units per milliliter standard and three cylinders with the 100 units per milliliter (estimated) sample, alternating standard and sample. A standard curve is prepared in the same manner as described for penicillin (Section 11,1), using standard concentrations of 180, 160, 140, 120, 100, 80, 60, 40, and 20 units per milliliter in 1% phosphate buffer, pH 6.0. Similarly, following the procedure of the penicillin assay, sample potencies are then calculated. 

Since it has been observed by the authors that modifications of the Rammelkamp broth dilution method for assay (28) enjoy the greatest popularity in laboratories today, a composite of these modifications, as used with satisfaction in the authors laboratory, is presented here. Practically all assay procedures for antibiotics discovered after penicillin are patterned on the penicillin assay itself. Therefore it serves our purpose to outline the assay for newer agents by indication of changes or substitutions which must be made in the basic (penicillin) method. 

Assay Medium.—Use the same milieu as employed in penicillin assay (Section IV, 1). 

Sterilise stainless steel penicillin assay cylinders and immerse in groups of 20 in 20 ml of a forty-eight-hour culture of Salm. cholercesuis or a twenty-four-hou. culture of Staph, aureus, transfer to a filter paper in a petn dish and dry in an incubator for not more than sixty minutes. Drop one cylinder into each of 10 tubes containing 10 ml of the chosen... 

By plate-diffusion The assay medium contains Oxoid peptone 10 g, Lab-Lemco 15 g and agar powder 15 g made up to 1 litre with water and adjusted to pH 7-2. For seeding the medium use about 0 4ml of a sensitive strain of Staphylococcus aureus (the Heat ley penicillin assay strain is suitable) grown for twenty-four hours in nutrient broth to each 100 ml of agar medium. 

ANHBIOHCS - BETA-LACTAMS - PENICILLINS AND OTHERS] (Vol 3) Assaying triarylmethane dyes... 

The reaction between 6-aminopenicillanic acid (6.5 g) and 3-o-chlorophenyl-5-methyllsoxa2ole-4-carbonyl chloride (7.66 g) gave the sodium salt of 3-o-chlorophenyl-5-methyl4-isoxa2olyl-penicillin (9.9Bg) asa pale yellow solid. Colorimetric assay with hydroxy lamina against a ben2-ylpenicillin standard indicated a purity of 6B%. 

The salt was prepared by dissolving the free acid form of the penicillin in the equivalent amount of aqueous sodium bicarbonate and freeze drying the resulting solution. The hydrated salt so obtained was shown by alkalimetric assay to be 94% pure and to contain 6% water. 

A suspension of 6-aminopenicillanic acid (36.4 grams) in water was adjusted to pH 7.2 by the addition of N aqueous sodium hydroxide and the resulting solution was treated with a solution of 3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carbonyl chloride (46.1 grams) in isobutyl methyl ketone. The mixture was stirred vigorously for hours and then filtered through Dicalite. The layers were separated and the isobutyl methyl ketone layer was shaken with saturated brine. Then, precipitation of the sodium salt only took place after dilution of the mixture with ether. In this way there was obtained 60.7 grams of the penicillin sodium salt having a purity of 88% as determined by alkalimetric assay. 

There was added to 250 ml of a concentrated butyl acetate extract containing 74,000 units of the acid form of penicillin per ml, 50 ml of a butyl acetate solution containing 0.238 g per ml of procaine base. The solution was agitated for one hour. The precipitate which formed was very gummy and not in the form of discrete crystals. This precipitate was crystallized by scratching the side of the vessel and agitating further. After this treatment 18.25 g of crystalline procaine penicillin was obtained which assayed 1010 units per mg representing a yield of 99.6% of the activity contained in the concentrated extract. 

Colorimetric assay with hydroxylamine showed this salt to contain 94% of the anhydrous penicillin. Paper chromatography showed complete reduction of the benzyl group. 

Drugs can also Interfere with laboratory results by negating certain nonspecific oxidation and reduction reactions essential for the chemical assay. Penicillin, streptomycin and ascorbic acid are known to react with cupric Ion thus, false positive results for glucose may occur If a copper reduction method Is used. If the specific enzymatic glucose-oxidase method Is employed, ascorbic acid can cause a false negative result by preventing the oxidation of a specific chromogen In the reaction. 

An immunoassay was developed to determine the penicillinase stable isoxazolyl penicillins cloxacillin and dicloxacillin in milk by Usleber et alJ The assay detected lOpgkg" of cloxacillin and 30pgkg of dicloxacillin with recoveries of 102% and 84%, respectively. The calibration curve was prepared by fortifying skimmed milk powder (lOOgL ) with standards. Fortified samples were prepared in pasteurized milk and analyzed directly after decreaming by centrifugation. This immunoassay was performed with minimal sample preparation, probably because the extensive water solubility of the penicillins prevents problems associated with more lipid-soluble analytes. 

The difference between the two titrations represents the volume of 0.02 N iodine equivalent to the total penicillins present in the given sample of benzylpenicillin. An assay may be carried out simultaneously by benzylpenicillin sodium (reference sample) so as to determine the exact equivalent of each ml of 0.02 N iodine. 

Disc Assay - This is the simplist of the procedures and involves the placing of a standard 1/2 disc saturated with milk onto the surface of B. stearothermophilus seeded agar plate and co-incubating with suitable control discs at 55 or 64 C until well-defined zones of inhibition are obtained, usually 3-4 h. Confirmation using penicillinase-treated milk is required. Zones 14.0 mm are positive. The lower limit of detection is 0.008 units penicillin/mL. This type of assay is simple, reasonably rapid and reasonably sensitive. Quantitation is possible by using graded concentrations of penicillin in the control milk. The technique is limited, however, to 3-lactam antibiotics, primarily penicillin ( ). 

A variation of the disc assay is the quantitative estimate using a central point. Each petri dish contains three reference discs which contain 0.016 units penicillin/mL and three discs saturated with the unknown milk. A penicillinase disc is placed in the center of each plate to help confirm the presence of penicillin. Three plates are used for each milk sample stearothermophilus is the assay organism. After incubation for 2-4 h, zones are measured and compared to the diameters of the reference concentration. Validity of the difference between zone size of the reference and sample is determined statistically. This procedure is less sensitive and attempts to set the qualitative presence of 3-lactams at 0.016 units/mL rather than at lower levels (3). 

Other miscellaneous assays for penicillin or other 3-lactams in milk is the Penzyme Test which uses cell wall enzjrmes inhibited by 3-lactam drugs in a kinetic assay. This test system is purported to be able to detect 0.005 units penicillin/mL and requires approximately 30 min to complete. It, like many other assays, detects 3-lactam antibiotics only. 

The BP utilises formation of a derivative in order to quantify penicillins in formulations. Some penicillins do not have distinctive chromophores a further problem with these molecules is that when they are in suspensions they are not readily extracted away from excipients since they are quite insoluble in organic solvents which are immiscible with water. Using the formation of a complex with the mercuric ion in the presence of imidazole as a catalyst, a derivative of the penicillin structure is produced, which has an absorption maximum between 325 and 345 nm. In the assay, comparison with pure standard for the particular penicillin is carried out rather than relying on a standard A(l%, 1 cm) value. This assay is used by the BP for... 

A mixture is to be assayed for penicillin. Added 10.4 mg ofpenicillin of specified activity 0.405 me... 

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