Amoxicillin analysis

Figure 1.9 [18] illustrates the use of CE for the analysis of amoxicillin (AMOX) from various suppliers. Unidentified impurities (1 and 2) and degradant (DG) are seen, and the mixtures are spiked with ampicillin (AMP) and penicillin V (PENV). 

Figure 1.9 Use of capillary electrophoresis for the analysis of amoxicillin from four suppliers (a-d). Reprinted from [18], copyright 1994, with permission from Elsevier. (AMOX is amoxicillin, AMP is ampicillin, PENV is penicillin V, DG is a degradant, and 1 and 2 are unspecified impurities. Capillary 80 cm X 50 (tm i.d. [75 cm to detector] [Polymicro Technologies] running buffer 100 mM pH 8 Na2HP04 containing 50 mM sodium dodecyl sulfate and 50 mM sodium borate voltage 18 kV injection at 100 mm for 15 s detector UV 205 nm.)...

It has been recently reported (109) that use of both Penase and lactamase II hydrolysis and screening assays prior to chromatographic analysis can tentatively classify -lactams into three subgroups the first group includes a ceftiofur metabolite represented by desfuroyl-ceftiofur-cysteine the second, cephapirin and the third, penicillin G, ampicillin, amoxicillin, and cloxacillin. In this approach, portions of aqueous extracts of tissues are treated separately with Penase and lactamase II, and results are compared with those of untreated samples and positive controls. Bioactive ceftiofur metabolites are present, provided that the extracts retain inhibitory activity after Penase treatment but lose activity after lactamase II treatment and are positive in response to the immunochemical Lac-Tek-Cef test but negative to the Lac-Tek-Bl test (113). This approach can eliminate a large number of negative samples and, therefore, increases the efficiency of the assay. 

A review of the chromatographic behavior of 90 penicillin and cephalspor-ins and their correlation to hydrophobicity has been published [339]. The current state of chromatographic methods submitted to the USP for com- plex antibiotics has been reviewed [340], A comparison has been made between poly(styrene-divinylbenzene) stationary phases and silica-based re-versed-phase sorbents for analysis of erthromycin and minocycline, and it was concluded that the nonsilica-based packings are more stable and ] reproducible [341]. The comparative retentions of ampicillin, amoxicillin,] and pencillin G was determined on Ci8, cyanopropyl-silica and poly(sty- rene-divinylbenzene), and nonbonded silica [342]. 

Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) of amoxicillin trihydrate showed loss of the water of crystallisation from about 50 to 150 °C followed by decomposition of the amoxicillin from about 160°C upwards [18], DSC and TGA of amoxicillin sodium salt in an oxygen atmosphere showed loss of water at about 100°C and thermal decomposition in multiple events from about 200 to 500°C [19]. The techniques are of little practical value for these compounds. 

Amoxicillin dimer, present as an impurity in amoxicillin sodium salt, was isolated and characterised as VII [SO], More detailed characterisation, together with data on amoxicillin trimer, was given in [48], Both compounds were isolated from an aged 20% solution of amoxicillin sodium salt, chromatographic analysis of which also indicated the presence of a tetramer and the penicilloates corresponding to these three oligomers. 

The main use of TLC in analysis of amoxicillin and its formulated products is as an identity test. A major study [105] using silica gel and silanised silica gel plates with thirty five different mobile phases and iodine vapour detection produced a system, on silanised plates with ammonium acetate/acetone mobile phase, in which amoxicillin was separated from all the other penicillins studied. A slightly modified version of this system was subsequently introduced into the European and British Pharmacopoeia monographs for amoxicillin trihydrate and sodium salt [2]. The British [57,58] and US [9] Pharmacopoeias specify other TLC systems for identification of amoxicillin in formulated products. Simple TLC methods have been developed for identification of several compounds on the WHO essential drugs list, to help combat counterfeiting [106]. For amoxicillin, systems using ethyl acetate/acetic acid/water or acetone/toluene/acetic acid/water with silica gel plates were recommended. 

Table 1 summarises HPLC methods which have been published for analysis of amoxicillin and its impurities and degradation products in the drug substances and formulated products. Most of these methods use the reverse phase mode on Cl8 columns, with UV detection and a mobile phase containing a small amount of methanol or acetonitrile in phosphate... 

For all APIs on the EML, it is imperative to consider not only the physical, chemical and absorption properties of the API when evaluating them for biowaiver, but (as outlined in the Multisource document ) to perform a benefit-risk analysis in view of the products usage at the national level. As an example, in some countries amoxicillin is used primarily for the treatment of ambulatory patients with mild-to-moderate infections of the upper respiratory tract, urinary tract and other sites. In other countries, amoxicillin might also be used to treat severe or even life-threatening infections, in which case the risk to the patient of arriving at the wrong bioequivalence decision would be far greater. 

Proton pump inhibitor-based three-drug regimens with two antibiotics (see Table 33-8) constitute first-line therapy for eradication of HP 1,5,36 meta-analysis of 666 studies indicates that PPI-based regimens that combine clarithromycin and amoxicillin, clarithromycin and metronidazole, or amoxicillin and metronidazole yield similar eradication rates (78.9% to 82.8%) nsing intent-to-treat analysis however, other studies suggest that the amoxiciUin-metronidazole combination is less effective. Eradication rates were improved when the... 

Erdmann, G.R. Walker, K. Giebink, G.S. Canafax, D.M. High performance liquid chromatographic analysis of amoxicillin in microliter volumes of chinchilla middle ear effusion and plasma. J.Liq.Chromatogr., 1990, 13, 3339-3350... 

Parker, C.E. Perkins, J.R. Tomer, K.B. Shida, Y O Haua, K. Nanoscale packed capillary liquid chro-matography-electrospray ionization mass spectrometry analysis of penicillins and cephems. J.Chromatogr, 1993, 616, 45-57 [serum also amoxicillin, ampicillin, carbenicillin, cefalothin, cefa-zohn, cefmenoxime, cefmetazole, cefoperazone, cefotaxime, cefotiam, cefoxitin, cloxacillin, dicloxacil-lin, penicillin G, pipericillin, sulbenicillin]... 

Drug analysis prints (Amoxicillin, claridiromycin, lansoprazole and metronidaa)le). Medicines and Healthcare Regulatory Agency, UK. Available at http //www.mhra.gov.uk/hcme/ idcplg IdcService=SS GET PAGE nodeId=742 (accessed 24/08/07). 

In visible spetrophotometric analysis of penam and cephem analogs, their ability to form ion-pair was also used. The penam analogs contacting the tertiary amine group (ampicillin, dicloxacillin, flucloxacillin, amoxicillin) and Mo(V)-thiocyanate binary complex in hydrochloric acid give coloured ion-pair formation absorbing at X = 467 nm (Fig. 14) [48]. 

A post-chromatographic derivatization method has been developed for penicillins, and was applied to the analysis of amoxicillin [119]. The compound was extracted from plasma and urine and separated by RP-HPLC with phos-plate buffer (pH 8.0) methanol (92 8, v/v) as the mobile phase. The derivatization was accomplished with an aqueous solution of imidazole (33%) and mercuric chloride (0.11%), pH 7.2, containing Brij 35 (0.12%). The derivative was detected at 310 nm. 

Antibiotics The AOAC has listed methods for sulfamethazine residues in swine tissues with determination either by GC-MS or GC-ECD of methylated derivatives and for sulfamethazine (and for the class of sulfonamides) in milk with determination by HPLC-UV. There is an AOAC method for the class of sulfonamide antimicrobials in animal tissues using solvent extraction and liquid partitioning with determination by TLC and fluorimetric scanning. For analysis of tetracyclines, AOAC describes methods based on buffer extraction from tissue samples and SPE (Cis) cleanup, or metal chelate affinity binding from milk samples, with determination in both cases by HPLC-UV. USDA/FSIS methods include (1) a method (similar to the AOAC GC-MS method for sulfamethazine) for confirmation of sulfonamide residues in edible tissues using solvent extraction and multiple liquid partitioning with determination of the methylated derivatives by GC-MS (2) methods for determination and confirmation of chloramphenicol in muscle by solvent extraction, liquid partitioning, and determination of the trimethylsilane (TMS) derivative by GC-ECD and GC-MS, respectively and (3) a method for determination of the beta-lactam antibiotic amoxicillin by aqueous extraction, cleanup by tricarboxylic acid precipitation, and ether extraction and formation of a fluorescent derivative for determination by LC. 

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