Chiral Separation of Antibiotics

The stereochemistry of an antibiotic is a prominent issue in the development, approval and clinical use of

Table 1 Determination of antibiotics by supercritical fluid chromatography

Column type

Sample

Comments

Detector Reference

Packed (1 mm i.d., 5 |im particles)Sulfonamides and and open tubular (50 |im i.d.) tetracyclines columns

Packed column 150 x 4.6 mm 5 |im particle amino silica

Packed column 100 x 4.6 mm 5 |im particle amino-bonded Spherisorb

Packed column 250 x 4.6 mm 10 |im particle Chiralcel OB

Open tubular 10 m x 50 ^m i.d. SB-biphenyl-100

Erythromycin A Cephalosporins

Sulfonamides, veterinary drugs

Lactams

1000C UV

8% Isopropanol as modifier

2-8% Methanol as modifier 75-90°C

15-25% Methanol as modifier

25oC

Chiral separation

Schmidt S, Blomberg, LG and Campbell ER (1988) Chromatographia 25: 775

MS UV Lane SJ (see Markides and Lee, 1988)

MS UV Perkins JR, Games DE, Startin JR and Gilbert JJ (1991) Journal of Chromatography 540: 239

Diode array Caude and Macaudiere (see Markides and Lee, 1988)

Cyclosporine A FK 506 70°C (Tacrolimus) Rapamycin Whole blood extracts

Open tubular 5 m x 50 ^m i.d. Macrolide antibiotic

DB-5

Different packed columns 250 x 4.6 mm 5 ^m particles

Midecamycin A1 Sulfonamides

Standard solutions

Great influence of temperature on resolution

Wong etal. (1994) Journal ofLiquid Chromatography 17: 2093

Ramsey et al. (1995) Analytical Proceedings 32: 455

Combs MT, Ashraf-Khorassani M and Taylor LT (1997) Journal of Chromatographic Science 35: 176

This summary is not intended to be a comprehensive review of all antibiotic separations by SFC; it aims to provide general information on the main applications in this field.

these drugs. For the separation of enantiomers, SFC with chiral stationary phases is very convenient due to its high resolution and relatively low analysis temperature.

Chiral separations in SFC can be carried out using open tubular columns with immobilized cyclodex-trins or, more recently, by packed columns with most of the same phases commonly used in LC, since the

Figure 3 Structures of some veterinary antibiotics analysed by SFC.

Figure 4 Chromatograms obtained from SFC of a mixture of veterinary antibiotics with UV (A, wavelength 215 nm; B, wavelength 230 nm). Peak identification: A, levamisole; B, furazolidone; C, chloramphenicol; D, lincomycin. Chromato-graphic conditions: column packed with 5 ^m particle amino-bonded Spherisorb (100x4.6 mm i.d.), column temperature 75°C, CO2 flow rate of 4mLmin~1, pressure 351 bar. Mobile phase was CO2 modified with 15% methanol. Reproduced with permission from Perkins etal. (1991).

Figure 4 Chromatograms obtained from SFC of a mixture of veterinary antibiotics with UV (A, wavelength 215 nm; B, wavelength 230 nm). Peak identification: A, levamisole; B, furazolidone; C, chloramphenicol; D, lincomycin. Chromato-graphic conditions: column packed with 5 ^m particle amino-bonded Spherisorb (100x4.6 mm i.d.), column temperature 75°C, CO2 flow rate of 4mLmin~1, pressure 351 bar. Mobile phase was CO2 modified with 15% methanol. Reproduced with permission from Perkins etal. (1991).

chiral selector is covalently bound to the packing material. The latter method frequently requires the addition of a modifier and is more common in the separation of drugs.

Several examples of the SFC of antibiotic enantio-mers can be found in the literature, although the technique is not as commonly used as LC. Packed columns with chiral stationary phases are normally employed with carbon dioxide modified with methanol or ethanol as mobile phase, under supercritical or subcritical conditions (i.e. at room temperature). Further applications can be expected from the use of packed capillary columns for chiral separations, which may provide better resolution and shorter analysis times than the equivalent LC separation.

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