Other Lactams

Epimers of phenethicillin (PEPC, Figure 3, structure 11), propicillin (PPPC, Figure 3, structure 12) and clometocillin are analysed with a Zorbax C8 column. The mobile phase is composed of methanol-water-5% 0.2molL~1 phosphate buffer (pH 7.0) and the epimers are detected at 254 nm. Ratios of methanol in the mobile phase are 37.5, 45 and 50% for PEPC, PPPC and clometocillin, respectively. Epi-mers are resolved close to the baseline, and the d-epimers elute faster than the corresponding l-epimers.

The same HPLC conditions can be used for the analysis of ampicillin, amoxicillin and azidocillin, except that the methanol content is varied between 10 and 40%. The l-epimer elutes faster for ampicillin, whereas the d-epimer elutes faster for amoxicillin and azidocillin. The less active epimers are not detected in the commercial preparations of these penicillins.

Epimers of PEPC and PPPC are also resolved with an ODS column. The mobile phase consists of 100 mmol L"1 ammonium acetate-methanol (62 : 38 and 58 : 42 for PEPC and PPPC, respectively) with a UV detection at 220 nm. PEPC and PPPC epimers are baseline separated (Figure 5).

Bacampicillin and cefotiam hexetil are the pro-drugs of ampicillin and cefotiam, respectively, which are commercially available as mixtures of two epi-mers due to chirality of the prodrug moiety. For separation of bacampicillin isomers, an ODS column is used with a mobile phase consisting of 20 mmol L"1 ammonium acetate-methanol (45 : 55). The isomers are detected at 220 nm. For separation of the isomers of cefotiam hexetil, an ODS column is used with a mobile phase consisting of 50 mmol L"1 phosphate buffer (pH 3.0)-acetonitrile (73 : 27). The isomers are detected at 262 nm. Baseline separation of the isomers of bacampicillin and cefotiam hexetil are observed.

Semisynthetic cephalosporins are extracted from biological fluids and chromatographed with an ODS column. Urine samples are merely centrifuged and diluted with distilled water. Serum samples are mixed with 0.4 molL"1 HCl and extracted with CHCl3-«-pentanol (3:1). The organic phase is re-extracted into phosphate buffer (pH 7), which is injected into the HPLC. The mobile phase is 0.01 mol L"1 acetate buffer (pH 4.8)-methanol (15:85) with detection wavelengths of 254, 245, 234, 275, 270, 240 and 240 nm for cefuroxime, cefoxitin, cefotaxime, cefazolin, cefamandole, cephalotin and cefoperazone, respectively.

Cephalosporins in serum are also analysed with an octyl column using a mobile phase of meth-anol-12.5 mmol L"1 phosphate buffer (pH 2.6, 1:4). Cefaclor, cefadroxil, cefixime, cephalexin and cephradine are simultaneously analysed and detected at 240 nm. The detection limits are 0.1 |ig mL"1 for cefixime and 1.0 igmL"1 for other cephalosporins. Serum is deproteinized with acetonitrile.

Cephalosporins with a tetrazole ring are analysed from plasma with an ODS column using a mobile phase consisting of 0.05 molL"1 phosphate buffer (pH 6.6)-methanol with ratios of 3 : 1 and 2 : 1 for cefamandole and cefoperazone, respectively. For cefotiam and cefmetazole, a mixture of phosphate buffer-tetrahydrofuran (20 : 1) is used as a mobile phase. Cephalosporins are detected at 254 nm with a limit of detection of 1 igmL"1 for all cephalos-porins.

In order to increase sensitivity, ampicillin, amoxicillin, cephalexin and cephradine in plasma are assayed after formation of fluorescent degradation products. Plasma is deproteinized with 10% trich-loroacetic acid and the supernatant is heated under various conditions to form degradation products. The degradation products are extracted with an organic solvent and injected into a Nucleosil C18 column at

Figure 5 Chromatogram of phenethicillin. One hundred microlitresof an aqueous solution of phenethicillin (22 ig mL 1) was directly injected onto HPLC.
Table 1 HPLC conditions for ß-lactams

ß-Lactam

Stationary phase

Mobile phase

Detection

Benzylpenicillin

ODS

Methanol-0.05 mol L~1 ammonium carbonate (1 : 3)

UV (254 nm)

Benzylpenicillin

ODS

Phosphate buffer (pH 6.0)-acetonitrile (4:1)

UV (225 nm)

Cefsulodin

ODS

16.8 mmol L~1 dibasic ammonium phosphate-acetic

UV (260 nm)

acid-methanol (100 : 1.68 : 5.98) containing 5 mmol L~1

triethylamine

Cefsulodin

ODS

Aqueous solution (containing 38.8 mmol L~1 ammonium

UV (260 nm)

acetate, 0.292 mmol L~1 dibasic ammonium phosphate

and 9.363 mmol L~1 triethylamine)-acetonitrile-

methanol-dimethylformamide-aceticacid

(1000:7.06:1.05:1.31 : 0.30)

Moxalactam

ODS

Methanol-0.05 mol L~1 monobasic potassium phosphate

UV (254 nm)

(5 : 95) adjusted to pH 6.5

Moxalactam

ODS

Methanol-0.005 mol L~1 tetran-butylammonium

UV (254 nm)

phosphate (1 : 3) adjusted to pH 6.0

Moxalactam

ODS

0.1 mol L~1 Ammonium acetate-acetonitrile (95 : 5)

UV (270 nm)

adjusted to pH 6.5

Moxalactam

ODS

0.1 mol L~1 Sodium phosphate-methanol (84 : 16)

UV (254 nm)

adjusted to pH 3.2

Temocillin

ODS

Methanol-0.1 mol L~1 phosphate buffer (pH 7.0, 1 : 9)

UV (230 nm)

Temocillin

Octyl silane

Methanol-0.1 mol L~1 phosphate buffer (pH 7.0, 16 : 84)

UV (230 nm)

7-Ureidoacetamido cephalosporins

ODS

0.01 mol L~1 Diammonium hydrogen phosphate

UV (254 nm)

containing 5-20% methanol containing 5-20% methanol

55°C. The mobile phase consists of methanol-water (3 : 2) with a fluorescent detection at 345 nm (excitation) and 420 nm (emission) for ampicillin, cephalexin and cephradine. For amoxicillin, the mobile phase is methanol-water (55 : 45) with a fluorescent detection at 355 nm (excitation) and 435 nm (emission). Detection limits are 0.5 ng mL_1 for ampicillin, 2ngmL~1 for cephalexin and 10ngmL_1 for amoxicillin and cephradine. For sensitive determination of ^-lactams, pre-column derivatization with imidazole-metal salt reagent or formaldehyde, or post-column derivatization with o-phthaldial-dehyde or fluorescamine may be applied.

HPLC conditions for several other ^-lactams are summarized in Table 1. Epimers of these ^-lactams are separated using the conditions listed in Table 1, except for benzylpenicillin.

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