Penicillin, assay standards

A casual examination of the current literature indicates that there is considerable confusion regarding antibiotic assay standards with regard to the terms unit and microgram used to indicate potency. Bacitracin and polymyxin are defined in terms of antimicrobial units which are not directly expressible in terms of weight of the compounds since these antibiotics have not been isolated in pure form penicillin originally fell into this category until its crystallization. For others which have been isolated in pure... 

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). 

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. 

To determine the potency of benzathine penicillin and of its preparations, the microbiological assay is employed using the Standard Preparation of Penicillin as standard. Because of the very low solubility of benzathine penicillin in water (about 1 in 6,000) a solution-aid is necessary and forma-mide or dimethylformamide is used. 

Benethamine Penicillin, Ci5Hi7N,CigHig04N2S, Mol. Wt. 545 7. Benethamine penicillin is the A -benzylphenethylamine salt of benzyl-penicillin and its potency is determined by microbiological assay (p, 814) using the Standard Preparation of Penicillin as standard. 

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%. 

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. 

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 ( ). 

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... 

Only free enzyme conjugate will bind to the coating. Addition of an enzyme substrate results in formation of a blue color, the intensity of which is inversely proportional to the amount of -lactam in the sample. Absence of -lactams in a sample results in all of the enzyme conjugate remaining unbound and available for binding to the immobilized -lactam. Presence of -lactams results in a portion of the enzyme conjugate being bound and unavailable to bind with the immobilized -lactam. The Delvo-X-Press test uses an optical density reader to compare the optical density of a standard set at a cutoff level of 5 ppb penicillin G, with that of each sample tube in total assay time of about 10 min. 

The experiment consists in the incubation of the labeled antigen (1) with increasing concentrations of MIP or NIP/CP, and (2) with the same set of concentrations but in the presence of a constant concentration of analyte sufficient to displace significantly the probe from the polymer [22]. Each point in the dilution curve should be determined at least by duplicate and preferably between five and ten replicates so that we can obtain not only the optimum polymer concentration for the assay but also an estimate of the standard deviation of the response at each point along the curves. The curve representing the difference (% bound label) of the bound label in the absence and in the presence of the analyte will render the optimum MIP concentration to be used in the assay. Using this approach we have optimized the amount of polymer required for the development of a fluoroimmuno-like assay for penicillin analysis [36],... 

An interesting example is die assay of clonidine hydrochloride in injections and tablets (British Pharmacopoeia, 1980). In 0.01 Mhydrochloric acid, clonidine exhibits two sharp maxima ne - 272 nm and 279 nm, which are not suitable for precise measurement. However, clonidine forms an ion pair with bromothymol blue, and this can be readily extracted into chloroform for subsequent measurement of the broad maximum near 420 nm. Because of the intiinsic variabihty of reagents used in such methods, a pharmacopoeial reference standard is employed for cahbration. A similar policy is adopted for assays involving chemical modification of the drug, as in the tetrazolium assay for corticosteroids, the assay for folic acid involving hydrolysis, diazotisation, and coupling with N-(l-naphthyl)ethylenediamine, and the reaction of penicillins with imidazole and mercuric salts. 

The classical iodometric assay of penicillins [S9] is applicable to amoxicillin and was the required procedure in the US Pharmacopoeia [60] prior to introduction of HPLC in 1991. This method is based on the fact that the intact penicillin nucleus does not react with iodine, but reaction does occur after hydrolysis to the penicilloic acid. Any penicilloic acid or other iodine reactive compounds present in the sample as impurities are corrected for by a blank titration of unhydrolysed penicillin. A slight variation of the standard procedure is required for amoxicillin and ampicillin in which a small amount of HC1 is added to the blank to release bound iodine which would otherwise cause false results [61], The reaction between iodine and penicilloic acid does not have an exact stoicheiometry and results are calculated relative to the purity of a reference standard which is assayed simultaneously with the sample. 

The cytotoxicity assay is usually performed by determining the viability of suitable cell lines in the presence of polymers. For this test, 3-5 mm discs of polymer film are cut and sterilised under standard conditions (at 121 C and 6.8 kg (15 lb) pressure for 15 min). The cell growth in the presence of the polymer films is measured under a controlled atmosphere (CO2 incubator, 37"C) using an appropriate culture medium, supplemented by 10% fetal bovine serum and penicillin-streptomycin antibiotic solution. Confluent monolayers are propagated by trypsinisation (0.25% trypsin and 0.02% EDTA, ethylene diamine tetraacetic acid) and re-plated at 2 x 10 cells/mL in a sterile polystyrene cell culture plate, then incubated for 24,48 and 72 h. The morphology of the cells is analysed by light naicroscopy (Leica) after... 

There are two generally accepted methods for the assay of streptomycin and dihydrostreptomycin. The plate diffusion assay uses a spore suspension inoculum and a technique almost identical to the penicillin plate assay described above. It has been the authors experience that the turbidimetric assay described below is more precise. This method is also applicable to dihydrostreptomycin with appropriate substitution of the standard. 

Assay Techniques. The general assay procedure is based on McMahan s turbidimetric assay of penicillin (27). The preparation of the standard curve and sample series is described below. 

A standard curve is prepared by diluting the standard solution to the following concentrations 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 /ig. per milliliter. Use three plates for the determination of each point on the curve. Filter paper discs (No. 740E, Schleicher Schuell, Keene, N. H.) serve as reservoirs for the antibiotic. The standard curve and sample calculations are the same as those used in the plate assay of penicillin (Section 11,1). 

Prepare a second series of twelve tubes by adding 0.5 ml. of the assay medium to all but 1. This rack may be labeled as the standard series. Deliver 0.5 ml. (i. e., 5 units) of penicillin standard solution to tubes 1 and 2. Mix the contents of tube 2 and prepare two-fold serial dilutions through tube 12, exactly as was done for the preparation of the sample dilution series." Tubes in the standard rack will then have the following penicillin concentrations (in units per tube) 6.0, 2.5,1.25,0.625, 0.312, 0.156,0.078, 0.039, 0.019, 0.009, 0.0045, and 0.0022. 

Assay Technique. Sample dilutions and standard dilutions may be prepared exactly as for penicillin (Section IV,1). By the addition of 0.6 ml. of standard streptomycin solution to the first two tubes in the series followed by dilution through tube 12, concentrations are obtained which range from 10.0 pg. in tube 1 to 0.0045 pg, in 12. 

Carry out the assay simultaneously using the Standard Preparation of penicillin to determine the exact equivalent of each ml of 0 02N iodine and from this calculate the result of the assav. 

It must be emphasised that this method is only suitable for the assay of benzylpenicillin in samples consisting substantially of benzylpenicillin. The assay should be repeated using the Standard Preparation of benzylpenicillin (dried crystalline sodium salt obtainable from the National Institute for Medical Research, assayed in terms of International Standard penicillin). The result should indicate that 95 8 per cent of the Standard is sodium benzylpenicillin if a lower value (but not less than 93 0 per cent) is obtained the assay may be considered valid and a proportionate correction should be applied to the result obtained on the sample under test. The purity of reagents is essential, particularly for the AT-ethylpiperidine. The temperature is important and should be ice cold whenever possible. If penicillin X (/)-hydroxy benzylpenicillin) is present the method may give an erroneous result, since a non-quantitative precipitation of the salt of penicillin X takes place. 

Preparations such as lozenges containing sugars as diluents yield erroneous avssays by the standard method. To overcome this, add either an equivalent amount of glucose to the standard penicillin solution or 0 5 per cent of glucose to the assay plate medium. 

From the result of the second assay (using standard) determine the amount of total penicillins, calculated as Ci6Hi704N2SNa, equivalent to each ml of 0-02N iodine, multiply it by 0-9832 to obtain the amount of total penicillins, calculated as C16H18O5N2S, equivalent to each ml of 0-02N iodine and from this calculate the result of the first assay. 

Although opinions vary concerning the absolute potency of pure benzyl-penicillin, on the evidence available it seems reasonable that for the purpose of this assay, the Standard Preparation of Penicillin containing 1,670 units... 

Microbiological methods are not used with the newer penicillins, except for their clinical estimation in blood, etc., because being mainly synthetic and of a difterent basic structure each possesses different antibacterial properties, hence each would require its own separate standard. The potencies of the new type penicillins cannot be properly related to those of benzylpenicillin for assay purposes. 

This compound became the standard of treatment for syphilis for over thirty years until it was phased out by other arsenicals and, finally, penicillin. Erlich s approach - create chemical diversity and assay for improved therapeutic properties - has changed little in the last century with the exception of the vast expansion of chemical space and the sophistication of the biological assays. 

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