Separation of Ceftibuten Stereo Isomers with 100% Aqueous Mobile Phase Using Zorbax SB-Aq Application

Drug Development
Adebayo O. Onigbinde

Ceftibuten dihydrate is currently one of the safest antimicrobial agents available to clinicians. As a result, it is one of the most frequently prescribed antibiotics. Ceftibuten belongs to the third generation of cephalosporin antibiotics (cephems), which are used to treat many varieties of infections caused by gram-positive and negative bacteria. Ceftibuten has two stereo isomers, the (Z) and the (E) isomers. Most HPLC solvents are considered hazardous to health and to the environment. Safe usage and disposal of large volumes of such solvents has always posed problems. Availability of columns, such as Zorbax SB-Aq, that can be used under high aqueous conditions and still retain consistent retention, resolution, selectivity, and peak shape is a great step in reducing the environmental impact of these solvents. Attempts to separate ceftibuten isomers on C18 columns with low percentages of organic modifiers (2%) did not give good resolution and peak shape (Figure 1). However, separation of ceftibuten isomers was achieved with 100% aqueous mobile phases on Zorbax SB-Aq column (Figure 2). Ceftibuten also exhibited very good linearity and a low limit of detection (LOD) (Figure 3).
DAD1 A, Sig = 254,100 Ref = 400,100 (71102C\001-0201.D)
mAU 300 250 200 150

Highlights
The Zorbax SB-Aq column has an alkyl bonded phase designed to eliminate phase collapse and still retain both hydrophilic and other compounds, when using highly aqueous mobile phases, including 100% water. Methods that require ion-pairing reagents for retention on other C8 or C18 bonded phases can often be carried out on the Zorbax SB-Aq column without ion-pairing reagents or organic modifiers. The Zorbax SB-Aq column separated ceftibuten stereo isomers with good peak shape and resolution in 100% aqueous phase without the use of ion-pairing agents or organic modifiers. The Zorbax SB-Aq column can deliver unique selectivity and resolution with very good peak shape for polar analytes that require low levels of organic modifier for adequate retention. The Zorbax SB-Aq column resolves compounds at a concentration commonly encountered in pharmacokinetic studies (Ceftibuten, LOD = 1.5 ng).

1.230 Excipent Z-Ceftibuten 1.371

1.064 1.151

100 50 0 0

E-Ceftibuten 2.578 5.015

6 7 8 9 min

Figure 1. Separation of ceftibuten isomers on C-18 columns with low percentages of organic modifiers. Concentration of ceftibuten dihydrate: 3.0 mg/mL.

Experimental Conditions
Instrument: Agilent 1100 Series HPLC; Temp: ambient; Column: Alkyl-C18, 4.6 150 mm, 5 m; Mobile phase: 2% ACN, 98% 10 mM ammonium acetate, pH 5.4; Flow rate: 1 mL/min; Injection volume: 5 L; Diode array detector: 254 nm; Reference: 400 nm; Bandwidth: 100 nm

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8.110
mAU 100 80 60
N O S H2N HO O H2N O H

DAD1 C, Sig = 254,4 Ref = 400,100 (51402B\001-0201.D)

OH O H H NH N N S O HO O S HO

Z-Ceftibuten

O H H NH N S

40 20

Excipent
0 0 2 4 6

E-Ceftibuten

10

12

13.477
14 min

Figure 2 Separation of ceftibuten isomers with 100% aqueous phases on Zorbax SB-Aq column. Concentration of ceftibuten dihydrate: 3.0 mg/mL.

Experimental Conditions
Instrument: Agilent 1100 Series HPLC; Temp: ambient; Column: Zorbax SB-Aq, 4.6 150 mm, 5 m (part number 883975-914); Mobile phase: 100% 10 mM ammonium acetate, pH 5.4; Flow rate: 1 mL/min; Injection volume: 5 L; Diode array detector: 254 nm; Reference: 400 nm; Bandwidth: 100 nm
250.00 200.00 Average area 150.00 100.00 50.00 0.00 0 5 10 Concentration (g/mL) 15 20

Ceftibuten

6.345

y = 13.86x - 0.6392 R2 = 0.9955

Adebayo Onigbinde is an applications chemist based at Agilent Technologies, Wilmington, Delaware, USA.

Figure 3. Good linearity and a low limit of detection for ceftibuten using a Zorbax SB-Ag column.

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Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc. 2002 Printed in the USA September 18, 2002 5988-7625EN