FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10; rule 13)
1. Title of the invention - "A process for the separation and detection of Penicillin
traces"
2. Applicant(s)
(a) NAME : ALEMBIC LIMITED
(b) NATIONALITY : An Indian Company
(c) ADDRESS : Alembic Campus, Alembic Road, Vadodara-390 003, Gujarat, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification describes the invention.

Field of Invention :
The present invention relates to a process for the separation and detection of Penicillin traces.
Particularly, the present invention relates to the use of a combination of two analytical methods to detect penicillin traces in bulk macrolide antibiotic like erythromycin thiocyanate.
Background of the invention and prior art:
The problem of penicillin hypersensitivity is well known to all engaged in the manufacturing of antibiotics.
Pharmaceutical drug manufacturers manufacturing Beta-lactams and other antibiotics, face the problem of cross contamination of drugs. This is a serious issue and has been addressed . by FDA in as early as 1965. Work was initiated to develop appropriate methods for the detection of residual penicillin as a contaminant in other antibiotics and non-antibiotic drug preparations.
Herbst et al (Oct 1965, Procedures for detecting and measuring penicillin contamination in Drugs Division of Antibiotics and Insulin certification. Food and Drug Administration (FDA), Washington D.C.) developed a simple chloroform extraction procedure which provided good recoveries of residual penicillin and ampicillin from erythromycin bulk and erythromycin stearate bulk. She also applied the' techniques of chromatography and bioautography method for detection of residual penicillin and ampicillin in demeclocycline and chlortetracycline antibiotics . Further development using a reverse phase TLC-bioautographic system led to a limit of detectability in the range of 0.5 - 1.0 ppm of penicillin in the tetracyclines (Doris Herbst, Journal of Pharmaceutical Sciences, Nov 1977).
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The US-FDA has laid down the strict requlatory requirements of not more than (NMT) 0.006 ppm traces of penicillin in any other antibiotics as a contaminant.
Therefore there was a need to develop a sensitive method / process with a high degree of repeatability and reproducibility which can detect such low levels of penicillin in other antibiotic drugs like erythromycin.
The inventors of the present invention have developed a simple, yet highly sensitive method for detecting as low as 0.006 ppm penicillin traces in erythromycin. Moreover the inventors believe that this method / process can also be applied for other antibiotics in the class of macrolide drugs and can be applied for detection of penicillin traces during decontamination of various processes of penicillin.
Summary of the invention
Therefore, it is an object of the present invention is to provide an improved method for the detection of traces (0.006ppm)of penicillin in erythromycin thiocyanate.
Another object is to provide an improved method for the separation and identification of penicillin traces in antibiotic bulk preparations.
Yet another object is to provide a method for separation and detection of traces of penicillin in bulk drug preparations which is simple, easy to perform, and highly sensitive.
Accordingly, the present invention provides a simple yet highly sensitive procedure of detection of contamination of residual penicillin in macrolide bulk preparation, using separation / analytical techniques of chromatography followed by bioautography.
The advantage of this technique is that it can detect concentration as low as 0.006 ppm which is an US-FDA requirement.
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The present invention can be also be applied for detecting penicillin contamination in a wide range of antibiotics which are hydrophobic in nature.
Detailed description of the invention:
The inventors of the present invention have found it possible to first separate the penicillin contaminant in the bulk antibiotic preparation and then detect the separated contaminant as penicillin. This two step analytical method provides a cent percent analysis of the nature of contaminant being as penicillin more so as penicillin-G.
The bulk erythromycin thiocyanate is extracted with aqueous solvents or buffers like cold water, buffers of salts of sodium or potassium phosphate, tris buffers, preferably water. The temperature of extraction is 15 - 28C preferably 22 - 25C. The pH of extracting solvent is from 5.5 to 7.0 preferably 6.0 to 6.5. Molarity of the buffer is in the range of 0.005 to 0.15M. The time of extraction depends on the quantity of antibiotic bulk, type of antibiotic bulk, quantity of extraction solvent and type of extracting solvent.
After extraction the extracted sample is subjected to TLC or RP-TLC or HPLC or HP-TLC. Solvent system comprising of water or acetone or acetone -water or acetone-buffer especially acetone - water in various ratios is used for separation.
After chromatographic separation the identification of the separated compounds is carried out by bioautogram.
The test indicator organism used could be either one of Staphylococcus aureus ATCC 9341, Sarcina lutea ATCC 15957, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 12692 and Bacillus subtilis ATCC 6633. However both S lutea ATCC 15957 or B subtilis ATCC 6633 are identified preferably to be highly sensitive to low doses of antibiotics.
The agar media employed for bioautogram mainly comprising of meat extract, beef extract, casein, dextrose, gelatin, sodium chloride, and peptone with a pH of 6.0 to 8.0 is used.
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After incubation of the agar plates at the desired time 7 to 10 hours and a temperature of 35 - 37°C for inhibition zones to appear, the zones of inhibition are read and marked clearly. Easy visualization of inhibition zone is possible using oxidation reduction dyes like methylene blue or TTC and zones corresponding to 0.006ppm penicillin is clearly detected (Enclosed is the standard bioautogram as Figure 1).
The following examples are meant to be illustrative of the present invention. These examples are presented to exemplify the invention and are not to be construed as limiting the invention's scope.
Example 1: Detection of 0.006ppm Penicillin
Standard USP penicillin G was taken. Stock solution was made in the range of 0.1 to 0.01 ppm in water or buffer of pH 6.0 to 6.5 at temperature of 22 to 25°C. This was spotted on TLC plate and chromatographed. The plate was then bioautographed. Clear zone of inhibition was seen upto 0.006ppm level.
Example 2: Penicillin traces in Erythromycin thiocyanate
The stock solution equivalent to 0.006ppm used in Example 1 was used as a standard for comparison. Erythromycin thiocyanate to be checked was taken and divided into two parts. To one part was added PenG to get a concentration of 0.006ppm. Both parts were then extracted either with water or buffer having a pH 6.0 to 6.5 at a temperature of 22 to 25°C. The extracted samples were spotted along with the standard of 0.006ppm on TLC plate and chromatographed. The plate was then subjected to bioautography. Clear zone of inhibition was seen in the standard and the spiked sample of erythromycin thiocyanate..
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and
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described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
AH patents, patent applications, and publications cited above are incorporated herein by reference in their entirety.

Dated this 31st day of March 2006.
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