synthesis, characterization and antibacterial …...impurity should be included. article received...

IJIPBART (2015) Volume 2, Issue (4), pp: 279-288 ISSN: 2349-865X

OPEN ACCESS

International Journal of Innovation in Pharma

Biosciences and Research Technology (IJIPBART)

Original Research Article

www.refsynjournals.com 279

Synthesis, characterization and antibacterial activities of

Clindamycin impurities

Ilango Kalpana1*, Mr. Blanchard Vincent

1

1 DEPARTEMENT CHIMIE, IUT D’ORSAY, UNIVERSITE PARIS SUD, FRANCE.

ABSTRACT

INTRODUCTION

Drugs are natural or synthetic substances used in the diagnosis, mitigation, treatment,

or prevention of a disease by functioning inside the living body. A legal or medicinal drug can be

harmful and addictive if misused. Impurities are nonessential compounds that are not part of the drug

substance but arise during the synthesis, extraction, purification and storage of the drug substance.

Process impurities are organic, inorganic and residual solvents which arise during the manufacturing

process of the drug substance.

Organic impurities arise during the manufacturing process and storage of new drug

substances. Inorganic impurities resulting from the manufacturing process can include reagents,

ligands and catalysts, heavy metals or other residual metals, inorganic salts and other materials.

Degradants are chemical break down compounds of drug substances formed during storage or

physical degradation. Degradants are unexpected adulterating compounds found in the drug substance

or different crystalline forms of the same drug substance. Many organic substances may exhibit

optical isomerism leading to the formation of enantiomers. Isomers, other than the drug substance are

treated as impurities. A general acceptance criterion of not more than 0.1% for any unspecified

impurity should be included.

Article received

December 11, 2015

Article accepted

December 21, 2015

Article published

December 31, 2015

*Corresponding Author:

Ms. Kalpana,

Departement chimie,

Universite paris sud,

France

[email protected]

Clindamycin is an antibiotic useful to treat a number of bacterial

infections. In this present study, the clindamycin impurities, namely

Sulphoxide and sulphone were synthesized by using low cost and

simple chemical mCPBA. The synthesized impurities were purified

by column chromatography. The formation of compounds were

identified by using TLC and HPLC. The compounds were

characterized by their respective spectral data (MS and 1H-NMR).

The antibacterial activity of impurities was compared against

clindamycin. The synthesized compounds should be beneficial for the

pharmaceutical, toxicological, clinical studies and for the drug

discovery and development.

Key words: clindamycin, sulphone, Sulphoxide, mCPBA, impurities

Ilango Kalpana et al IJIPBART (2015) Volume 2, Issue (4), pp: 279-288


Clindamycin is semi-synthetic and belongs to lincosamide class (Brodasky et al., 1968).

Clindamycin is a chlorine substituted derivative of lincomycin (trans-L-4-n-propyl hygrinic acid,

attached to a sulfur containing derivative of octose), synthesised by Streptomyces lincolnensis.

Clindamycin is active against B. fragilis, P. jiroveci, T. gondii, Clostridium, C. acnes and P. carinii.

So, it is used in the treatment of abdominal and pelvic abscess, respiratory tract infections including

lung abscess, skin and soft-tissue infections. Clindamycin is usually similar to erythromycin in its in

vitro activity against susceptible strains of Pneumococci, S. pyogenes, and S. viridans. Methicillin

susceptible S. aureus strains are susceptible to Clindamycin whereas methicillin resistant S. aureus

strains and coagulase negative Staphylococci are resistant to Clindamycin. Clindamycin is more active

against anaerobic bacteria, especially, B. fragilis, than erythromycin or clarithromycin. Other

Bacteroides species and anaerobes like B. melaninogenicus, Peptostreptococcus, Peptococcus and C.

perfringens are susceptible to Clindamycin. Actinomyces israelii, Nocardia asteroides and

Chlamydia species are sensitive to Clindamycin.

Clindamycin is used against protozoans such as Toxoplasma and Mycoplasma as well as

many anaerobic bacteria (Luft and Remington, 1988; Dannemann et al., 1991; Mazur et al., 1999). In

humans, absorption of clindamycin is rapid and virtually complete (90%) following oral

administration (DeHaan et al., 1972; Metzler et al., 1973). Concentration of Clindamycin in the serum

increases linearly with increased dose, and the levels exceed the minimum inhibitory concentration

for most indicated organisms for atleast 6 hours following administration of the recommended dose.

Clindamycin is widely distributed throughout the body and has an average biological half life of 2.4

hours. The major bioactive metabolites excreted in urine and faeces are Clindamycin sulfoxide and N-

desmethylclindamycin (Seaberg et al., 1984; Flaherty et al., 1988; Gatti et al., 1998). The synthesis of

Clindamycin sulphone and sulphoxide was done using sodium perborate and Hydrogen peroxide

(Devlin, 2006). Microbiological synthesis of Clindamycin sulphoxide has also been reported

(Arcoudellis et al., 1969).

Oxidation is the process of converting materials into useful chemicals of a higher oxidation

state (Hudlicky, 1990). Nitric acid, the most conventional industrial oxidant, (Buchner et al., 1986) is

cheap but forms various nitrogen oxides. Oxidation of organic sulfides is the most utilized method to

produce sulfoxides and sulfones (Patai et al., 1988; Patai and Rappoport, 1994; Page, 1995; Kagan,

1998). This method is also useful to study the oxygenation properties of oxidation systems (Ricoux,

2009; Franke, 2008). Stoichiometric oxidants like permanganate (Gokel et al., 1998), sodium bromate

(Shaabani et al., 2009), periodic acid (Xu et al., 2003), perbenzoic acid (Paybarah et al., 1982), meta-

chloroperbenzoic acid (Kubota and Takeuchi, 2004) and sulfonic peracids (Kluge et al., 1996) have

also been used. Oxidation reagents play a crucial role in the organic synthesis and the most important

among them is meta-choroperoxybenzoic acid (mCPBA). Advantages of 3-chloroperbenzoic acid is

its handling, because it is present as powder, which can be kept in the refrigerator. mCPBA with an

outstanding reactivity is however, more selective than hydrogen peroxide and other peracids.



In the present study, mCBPA was used in simple environmental conditions to synthesize

Clindamycin Sulphoxide and Clindamycin sulphone. Thus, objective of the present study is the

synthesis and characterization of Clindamycin Sulphoxide and Clindamycin sulphone impurities by

using mCPBA.

METHODS

Clindamycin was gifted by Refsyn Biosciences Pvt. Ltd, Puducherry. All reagents and

solvents used were of commercial grade and were used as such, unless otherwise specified. Thin

Layer Chromatography (TLC) was performed on Kieselgel 60 F254 silica-coated aluminium plates

(Merck, India) and visualized using iodine. Organic extracts were dried over anhydrous Sodium

sulphate.

Synthesis of Clindamycin sulphoxide

1g of Clindamycin was dissolved in 4 ml of acetic acid and cooled to 0 °C mCPBA was added

and the reaction was continued by heating to 50 °C. The reaction was monitored by Thin layer

chromatogram and visualized under iodine chamber. Completion of the reaction was observed by the

disappearance of Clindamycin. The reaction mixture was neutralized to PH

10 using saturated sodium

carbonate. The compound was extracted with chloroform. Acetone was added to the aqueous layer.

The precipitated white solid was filtered and washed with diethyl ether and dried. The crude material

was purified by column chromatography using silica gel 60-120 mesh and Chloroform and methanol

mixture as mobile phase.

Synthesis of Clindamycin sulphone

250 mg of Clindamycin was dissolved in 10 ml of methanol. To this, 1.5 equivalents of

mCPBA were added and the reaction was continued at room temperature. The reaction was monitored

by Thin layer chromatogram and visualized under iodine chamber. Completion of the reaction was

observed by the disappearance of Clindamycin. The reaction mixture was neutralized to PH

8 using

saturated sodium carbonate. The compound was extracted with chloroform. The organic layer was

taken and dried with anhydrous sodium sulphate. The crude material was purified by column

chromatography using silica gel 60-120 mesh and Chloroform and methanol mixture was used as

mobile phase.

TLC analysis of crude Clindamycin sulphone and sulphoxide compounds

Clindamycin, Clindamycin sulphoxide and Clindamycin sulphone were analyzed by TLC to

determine the Rf values. 20% methanol in chloroform was used as mobile phase and visualized under

iodine chamber.

HPLC analysis of crude Clindamycin sulphone and sulphoxide compounds

Clindamycin, Clindamycin sulphoxide and Clindamycin sulphone were analyzed using an

isocratic HPLC system (Waters, USA) to determine the conversion of products. Buffer solution was

prepared by dissolving 6.8 g of monobasic potassium phosphate in 1 litre of water and adjusting the



PH to 7.5 with 8N Potassium hydroxide. The buffer was filtered through 0.45 µm filter. 55% buffer

solution was mixed with 45% acetonitrile, followed by ultrasonication for 15 min. Mobile phase was

filtered through 0.45 µm filter. The samples were dissolved in methanol and filtered through 0.45 µm

filter and used for HPLC analysis.

Characterization of the purified compounds

Molecular weights of the purified Clindamycin sulphoxide and Clindamycin sulphone

compounds were analyzed by Mass Spectrometry and structural identification was performed by H1

NMR spectroscopy. NMR (Nuclear Magnetic Resonance) spectra was recorded using a Spectrometer

(Varian 300 MHz Mercury plus) at 300 MHz. Chemical shifts were given in ppm relative to

trimethylsilane (TMS). Mass spectra were recorded on Shimadzu-LC-2010EV Liquid

Chromatography-Mass Spectrometry with APCI and ESI probes (Shimadzu, Japan).

Testing the antibacterial activity of the purified compounds

The antibacterial activity of the purified compounds was tested by well difusion method on

Muller Hinton agar plates (Himedia Laboratories, Mumbai, India). Test solution of concentration 93

mg/ml was used for the analysis. Overnight bacterial cultures of Staphylococcus aureus NCIM 2079,

Streptococcus mutans NCIM 2611, Bacillus subtilis NCIM 2920, Enterobacter faecalis NCIM 2015,

Escherichia coli NCIM 2065 and Klebsiella pneumoniae NCIM 2957 were used as inoculum for

testing the antibacterial activity. 50µl of Clindamycin sulfoxide and Clindamycin sulfone impurities

were added to the wells and incubated and the results were recorded by measuring the zone of

inhibition using calibrated reader scale.

RESULTS AND DISCUSSION

The synthesis reaction of Clindamycin sulphone and Sulphoxide were formed in 6 & 12 hours

respectively. The sulphone was extracted ethyl acetate and the Sulphoxide present in aqueous which is

precipitated with acetone (Figure 1).

TLC and HPLC are best methods for the identification of compounds. The reaction was

monitored by using TLC and was observed in the iodine chamber due to inactivation of clindamycin

in UV Light (Figure 2). The yield of white solid Sulphoxide and sulphone were 60% & 90%.

HPLC analysis is used for the identification of pharmaceutical products like metabolites and

impurities. Less volume of samples are sufficient for HPLC analysis. The conversion of products can

be easily detected by HPLC. HPLC analysis of Clindamycin, crude compounds of Clindamycin

sulphoxide and Clindamycin sulphone were done by isocratic method. Retention Time (RT) of

Clindamycin, Clindamycin sulphoxide and Clindamycin sulphone were 6, 4 and 3.5 respectively

(Figure 3). These results show the formation of products with some unknown impurities. The

conversion percentage of sulphoxide and sulphone were 61% and 92% respectively.

Ilango Kalpana et al

www.refsynjournals.com

Figure-1: Extraction (A), crude compound (B), column purification and product of Clindamycin

Figure 2: TLC analysis of pure Clindamycin sulphoxide and Clindamycin sulphone

Purification of Clindamycin, Clindamycin

Figure-3: HPLC analysis images of Clindamycin

sulphone (C)

Table-2: HPLC analysis result of Clindamycin

Sl. No. Name RT

1 Clindamycin 6.45

Sum

IJIPBART (2015) Volume 2, Issue (

Extraction (A), crude compound (B), column purification and product of Clindamycin

sulphoxide & Sulphone

Table-1: Rf values of compounds

TLC analysis of pure Clindamycin sulphoxide and Clindamycin sulphone

Purification of Clindamycin, Clindamycin Sulphoxide and Clindamycin Sulphone

HPLC analysis images of Clindamycin(A), crude Clindamycin Sulphoxide (B) &

HPLC analysis result of Clindamycin

RT [min] Area [mVxs] Height [mV] Amount %

6.45 2184.24 69.59 100

2184.24 69.59

Name of the compound Rf values

Clindamycin 0.94

Clindamycin sulphoxide 0.20

Clindamycin sulphone 0.86

IJIPBART (2015) Volume 2, Issue (4), pp: 279-288

283

Extraction (A), crude compound (B), column purification and product of Clindamycin

TLC analysis of pure Clindamycin sulphoxide and Clindamycin sulphone

Sulphoxide (B) &

Amount %

100



Table-3: HPLC analysis result of crude Clindamycin sulphoxide

Sl. No. Name

1. Unknown 1

2. Clindamycin Sulfone

3. Clindamycin Sulfoxide

4. Unknown 2

Sum

Table-4: HPLC analysis result of crude Clindamycin sulphone

Sl. No. Name

1. Unknown

2. Clindamycin Sulfone

Sum

Characterization of compounds

Figure-4: Mass Spectrometry and H

and B) and Clindamycin sulphone (C and D)


HPLC analysis result of crude Clindamycin sulphoxide

RT [min] Area [mVxs] Height [mV]

2.85 892.06 37.33

3.5 417.88 21.86

4.08 814.70 44.36

4.88 100.70 5.02

2225.34 108.57

HPLC analysis result of crude Clindamycin sulphone

RT [min] Area [mVxs] Height [mV]

2.83 1056.64 39.61

3.42 1174.36 113.20

2231.01 152.81

Mass Spectrometry and H1 NMR Spectroscopy analysis of Clindamycin sulphoxide (A

and B) and Clindamycin sulphone (C and D)


284

Amount %

26

10

61

3

Amount %

8

92

NMR Spectroscopy analysis of Clindamycin sulphoxide (A



The mass spectrometry and H

Clindamycin sulphoxide and Clindamycin sulphone. By using atmospheric pressur

ionization, MS spectra were produced consisting predominantly of the protonated [M+H]

precursor ions with a mass to charge ratio (m/z) of

charge ratio (m/z) of 456 implied Clindamycin su

and stereochemistry of the molecules of pharmaceutical interest can be

H1 NMR spectroscopy analysis was performed for the ide

Antibacterial activity of Clindamycin and its impurities

Figure-5: Antibacterial activity of Clindamycin (A) and its impurities (B & C) in different

Table-5: Zones of Inhibition for the bacterial cultures

Bacteria

Staphylococcus aureus

Streptococcus mutans

Enterobacter faecalis

Bacillus subtilis

Klebsiella pneumoniae

Escherichia coli

The bacteria used for the study showed a significant antibacterial effect against both the

impurities towards S. mutans, S. aureus

Similarly, for K. pneumonia, Clindamycin S

Clindamycin Sulphoxide. The impurities exhibited bacteriostatic activity against

minimum incubation period proportional to the concentration and as the incubation period increases

the bacteria dominated by increasing its population size with an indication that the drug impurity

possesses bacteriostatic effect thus not completely killing


The mass spectrometry and H1 NMR spectroscopy results confirm the formation of

Clindamycin sulphoxide and Clindamycin sulphone. By using atmospheric pressur

ionization, MS spectra were produced consisting predominantly of the protonated [M+H]

a mass to charge ratio (m/z) of 440 implied Clindamyicn sulphoxide and a mass to

456 implied Clindamycin sulphone (Figure 4). The specific bonding structure

and stereochemistry of the molecules of pharmaceutical interest can be studied by NMR

analysis was performed for the identification of protons (Figure 4

activity of Clindamycin and its impurities

Antibacterial activity of Clindamycin (A) and its impurities (B & C) in different

bacterial cultures

Zones of Inhibition for the bacterial cultures

Clindamycin Clindamycin

Sulphone

Clindamycin

Sulphoxide

Staphylococcus aureus 40 mm 20 mm 24 mm

40 mm 28 mm 30 mm

40 mm 19 mm 18 mm

40 mm 30 mm 32 mm

Klebsiella pneumoniae 44 mm 27 mm 16 mm

22 mm No Zone No Zone


S. aureus and B. subtilis with a inhibition zone as noted in Table 5.

Clindamycin Sulphone exhibited better bactericidal activity than

. The impurities exhibited bacteriostatic activity against E. coli


inated by increasing its population size with an indication that the drug impurity

possesses bacteriostatic effect thus not completely killing E. coli.


285

the formation of

Clindamycin sulphoxide and Clindamycin sulphone. By using atmospheric pressure chemical

ionization, MS spectra were produced consisting predominantly of the protonated [M+H]+ ion. The

lindamyicn sulphoxide and a mass to

). The specific bonding structure

by NMR spectroscopy.

ntification of protons (Figure 4).

Antibacterial activity of Clindamycin (A) and its impurities (B & C) in different

Clindamycin

Sulphoxide


with a inhibition zone as noted in Table 5.

exhibited better bactericidal activity than

E. coli in the


inated by increasing its population size with an indication that the drug impurity



CONCLUSION

Clindamycin sulphoxide and sulphone compounds were prepared by using mCPBA with

different equivalents and with different temperatures. The crude compounds were analysed by TLC

and by HPLC to determine the conversion rate of compounds. The compounds were purified by

column chromatography. The purified compounds were analysed by Mass Spectrometry and H1 NMR

spectroscopy. The antibacterial activities of Clindamycin sulphoxide and Clindamycin sulphone were

compared with that of Clindamycin. mCPBA is the very less expensive and a strong oxidising agent

used for the sulfoxidation of macrolide Clindamycin. The synthesized compounds were also having

higher antibacterial activity compared to Clindamycin. So the impurities can also be used in drug

discovery, drug developmental studies and also as reference standards for phamacological,

toxicological and clinical trial studies.

REFERENCES

1. Brodasky, T.F., Argoudelis, A.D., and Eble, T.E. 1968. The characterization and thin-layer

chromatographic quantitation of the human metabolite of 7-deoxy-7(S) chlorolincomycin (U-

21,251F). J Antibiot (Tokyo) 51: 327-333.

2. Hudlicky, M. 1990. Oxidations in Organic Chemistry, ACS Monograph Ser. 186, American

Chemical Society, Washington, DC (b) Comprehensive Organic Synthesis, ed. B.M. Trost and I.

Fleming, Pergamon Press, Oxford, 1st edn., 1991,7.

3. Buchner, W., Schliebs, R., Winter, G., and Buchel, K.H. 1986. Industrielle Anorganische Chemie,

VCH, Weinheim, Germany, 2nd edn.

4. Patai, S., Rappoport, Z., and Stirling, C.J.M. 1988. The chemistry of sulphones and sulphoxides.

Wiley, New York.

5. Patai, S., and Rappoport, Z. 1994. The synthesis of sulphones, sulphoxides and cyclic sulfides.

Wiley, New York.

6. Page, P.C.B. 1995. Organosulfur chemistry: synthetic aspects. Academic Press, London

7. Kagan, H.B., and Luukas, T. 1998. In: Beller M, Bolm C (eds) Transition metals for organic

synthesis, 2nd edn. Wiley, Weinheim, pp 361-373.

8. Ricoux, R., Allard, Dubuc M, Dupont C, Marechal JD, Mahy J.P. 2009. Selective oxidation of

aromatic sulfide catalyzed by an artificial metalloenzyme: new activity of hemozymes. Org

Biomol Chem 7: 3208-3211.

9. Franke, A., Fertinger, C., and Van Eldik, R. 2008. Angew Chem Int Ed 47:5238-5242.

10. Gokel, G.W., Gerdes, H.M., and Dishong, D.M. 1980. Sulfur heterocycles. 3. Heterogeneous,

phase-transfer, and acid-catalyzed potassium permanganate oxidation of sulfides to sulfones and a

survey of their carbon-13 nuclear magnetic resonance spectra. J Org Chem 45: 3634-3639.

11. Shaabani, A., Behnam, M, and Rezayan, A.H. 2009. Tungstophosphoric acid (H3PW12O40)

catalyzed oxidation of orga.nic compounds with NaBrO3. Catal Commun 10: 1074-1078.



12. Xu, L., Cheng, J., and Trudell, M.L. 2003. Chromium (VI) oxide catalyzed oxidation of sulfides

to sulfones with periodic acid. J Org Chem 68: 5388-5391.

13. Paybarah, A., Bone, R.L., and Corcoran, W.H. 1982. Selective oxidation of dibenzothiophene by

peroxybenzoic acid formed in situ. Ind Eng Chem Process Des Dev 21: 426-431.

14. Kubota, A., and Takeuchi, H. 2004. An unexpected incident with m-CPBA. Org Process Res Dev

8: 1076-1078.

15. Kluge, R., Schulz, M., and Liebsch, S. 1996. Sulfonic peracids- III. Heteroatom oxidation and

chemoselectivity. Tetrahedron 52: 5773-5782.

16. Luft, B.J., and Remington, J.S. 1988. Toxoplasmic encephalitis. J Infect Dis 157: 1-6.

17. Dannemann, B.R., McCutchan, J.A., Israelski, D.M., Antoniskis, D., Leport, C., Luft, B.J., Chiu,

J., Vilde, J.L., Nussbaum, J.N., Orellana, M., Heseltine, P.N.C., Leedom, J.M., Clumeck, N.,

Morlat, P., Remington, J. S. and the California Collaborative Treatment Group. 1991. Treatment

of acute toxoplasmosis with intravenous Clindamycin. The California Collaborative Treatment

Group. Eur J Clin Microbiol Infect Dis 10: 193-195.

18. Mazur, D., Schug, B.S., Evers, G., Larsimont, V., Fieger-Buschges, H., Gimbel, W., Keilbach-

Bermann, A., and Blume, H.H. 1999. Bioavailability and selected pharmacokinetic parameters of

clindamycin hydrochloride after administration of a new 600 mg tablet formulation. Int J Clin

Pharmacol Ther 37: 386-392.

19. DeHaan, R.M., Metzler, C.M., Schellenberg, D., VandenBosch, W.D., and Masson, E.L. 1972.

Pharmacokinetic studies of clindamycin hydrochloride in humans. Int J Clin Pharmacol 6: 105-

119.

20. Metzler, C.M., DeHaan, R., Schellenberg, D., and Vandenbosch, W.D. 1973. Clindamycin dose

bioavailability relationships. J Pharm Sci 62: 591-598.

21. Seaberg, L.S., Parquette, A.R., Gluzman, I.Y., Phillips, G.W., Brodasky, T.F., and Krogstad, D.J.

1984. Clindamycin activity against chloroquine- resistant Plasmodium falciparum. J Infect Dis

150: 904-911.

22. Flaherty, J.F., Rodondi, L.C., Guglielmo, B.J., Fleishaker, J.C., Townsend, R.J., and

Gambertoglio, J.G. 1988. Comparative pharmacokinetics and serum inhibitory activity of

clindamycin in different dosing regimens. Antimicrob Agents Chemother 32: 1825-1829.

23. Gatti, G., Malena, M., Casazza, R., Borin, M., Bassetti, M., and Cruciani, M. 1998. Penetration of

clindamycin and its metabolite N-desmethylclindamycin into cerebrospinal fluid following

intravenous infusion of clindamycin phosphate in patients with AIDS. Antimicrob Agents

Chemother 42: 3014-3017.

24. Devlin, J.W., Fong, J., Schumaker, G., Ruthazer, R., o'connor, H., and Garpestad, E. 2006. Use of

a validated delirium assessment tool improves the ability of physicians to recognize delirium in

medical intensive care unit (MICU) patients.: 22. Crit Care Med 34(12) : p A6

25. Arcoudellis, A.D., Coats, J.H., and Sebek, O.K. 1969. Microbial transformation of Antibiotics.



III. Conversion of clindamycin to 1’-demethylclindamycin and clindamycin sulfoxide by

Streptomyces species. J Antibiot 22: 309-314.

26. Thomas, L.A., T Bizikova, and AC Minihan, In vitro elution and antibacterial activity of

Clindamycin, Amikacin, and Vancomycin from R-gel Polymer, DVM, Diplomate ACVS,

Chesapeake Veterinary Surgical Specialists, Annapolis, MD and Royer Animal Health, LLC,

Frederick, MD, by The American College of Veterinary Surgeons,2011.

Cite this article in press as Ilango Kalpana et al. (2015) Synthesis, characterization and

antibacterial activities of Clindamycin impurities, IJIPBART, 2(04); 279-288.

CONFLICTCONFLICTCONFLICTCONFLICT OF INTERESTSOF INTERESTSOF INTERESTSOF INTERESTS

The authors declare that they have no conflict of interests regarding the publication of this paper.

Copyright © 2015 by authors. This is an open access article distributed under the Creative Commons Attribution License, which

permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

synthesis, characterization and antibacterial …...impurity should be included. article received...

Documents