Analysis of Antibacterial Drugs by HPLC

Udo Huber Pharmaceutical

Abstract This Application Brief describes HPLC analyses of three groups of antibacterial drugs: penicillins tetracyclines other antibacterial drugs Penicillins Penicillins can be isolated from the culture medium of certain fungi, producing natural penicillin, for example, Penicillium notatum or P. chrysogenum. Other penicillins can be synthesized semisynthetically or by precursor-indi-cated biosynthesis. Total synthesis would not be economical. Penicillin inhibits the polymerization of murin, which is responsible for the stability of the bacteria's cell wall. Figure 1 shows the HPLC separation of four common antibacterial drugs with pencillin-like structure (amoxicillin, ampicillin, penicillin G and penicillin V). Gradient analysis on a reversed phase column and UV detection were used. The autosampler temperature was set to 4 C to avoid decomposition of the samples.

Conditions

Absorbance [mAU] 1 2 3 4 Amoxicillin Ampicillin Penicillin G Penicillin V

1000 800 600 400 200 0 0 2 4 6 Time [min] 8 1 4 2 3

10

12

Figure 1 EIC traces from amine standards

Column 4.6 x 75 mm Zorbax SB-C18 3.5 m Mobile phase A = 0.025 M KH2PO4 in water (pH = 3 with H2SO4) B = acetonitrile Flow rate 1.0 ml/min Gradient 5 % B to 60 % B in 10 min Column wash 60 % B to 5 % B in 2 min UV detector variable wavelength detector 204 nm, standard cell Column compartment temperature 40 C Stop time 12 min Post time 5 min Injection volume 5 l

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Table 1 shows the performance of the HPLC method for these four pencillin drugs. Compound LOD for S/N=2 (mg/l)* Precision of RT (RSD of 10 runs) (100 mg/l)* 0.07 0.07 0.03 0.02 Precision of Area (RSD of 10 runs) (100 mg/l)* 0.69 0.57 0.41 1.06 Tetracyclines Some tetracyclines are natural compounds from different streptomyces species. Besides being used in human and veterinary medicine they are fed as nutritional antibiotics in pig and poultry farming. Because of their long half-life and resistance this is not allowed in Germany.

Ampicillin 1.0 Amoxicillin 1.0 Penicillin G 1.0 Penicillin V 1.0 * Injection volume: 5l

Table 1 HPLC method performance of antibacterial drugs with penicillin-like structure

Figure 2 shows the chromatogram of three antibacterial drugs with tetracyclic structure using gradient analysis on a reversed phase column and UV detection. To avoid decomposition of samples the autosampler temperature was set to 4 C. Table 2 shows the performance of the HPLC method for the analyzed tetracyclines.
Absorbance [mAU] 2 120 100 80 60 40 20 0 0 2 4 Time [min] 6 8 10 1 3 1 Minocycline 2 Tetracycline 3 Doxycycline

Conditions
Column 4.6x75 mm Zorbax SB-C18 3.5 m Mobile phase A = 0.025 M KH2PO4 in water (pH = 3 with H2SO4) B = acetonitrile Flow rate 1.0 ml/min Gradient 5 % B to 60 % B in 10 min Column wash 60 % B to 5 % B in 2 min UV detector variable wavelength detector 350 nm, standard cell Column compartment temperature 25 C Stop time 12 min Post time 5 min Injection volume 5 l

Figure 2 Separation of three antibacterial drugs with tetracyclic structure Compound LOD for S/N=2 Precision of RT (mg/l)* (RSD of 10 runs) (100 mg/l)* 0.06 0.05 0.04 Precision of Area (RSD of 10 runs) (100 mg/l)* 0.14 0.13 0.21

Minocycline 0.1 Tetracycline 0.1 Doxycycline 0.1 * Injection volume: 5 l

Table 2 HPLC method performance of antibacterial drugs with tetracyclic structure

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Other Antibacterial Drugs Nalidixic acid and the combination of the synthetic compounds trimethoprim and sulfamethoxazole are especially effective in treating infections of the urinary tract. Chloramphenicol is a natural compound found in streptomyces. It can also be synthesized easily. Chloramphenicol is used in treating a wide range of bacterial diseases, for example, typhoid or parrot fever. Furazolidone is used mainly against trichomonades and intestinal bacteria. Figure 3 shows the chromatogram of six antibacterial drugs using gradient analysis on a reversed phase column and UV detection. To avoid decomposition of samples the autosampler temperature was set to 4 C.
Absorbance [mAU] 400 350 300 250 200 150 100 50 0 0 2 4 6 Time [min] 8 10 12 14 3 5 2 4 6 1 204 nm 4.5 min 368 nm 1.5 min

Conditions
204 nm 4 min 254 nm 5 min 204 nm 1 Hydroxybenzotriazole 2 Trimethoprim 3 Furazolidone 4 Sulfamethoxazole 5 Chloramphenicol 6 Nalidixic acid

Figure 3 Analysis of six antibacterial drugs using a variable wavelength detector The analysis of trimethoprim and sulfamethoxazole can also be done using a fluorescence detector as shown in figure 4.
Excitation 230 nm 275 nm 343 nm Emission 503 nm

Emission [LU]

230/503 nm

20 15 10 5 Trimethoprim 200 250 300 350 400 450 Wavelength [nm] Sulfamethoxazole

Trimethoprim 20

0 500

Column 4.6x75 mm, Zorbax SB-C18 3.5 m Mobile phase A = 0.025 M KH2PO4 in water (pH = 3 with H2SO4) B = acetonitrile Flow rate 1.0 ml/min Gradient 10 % B to 30 % B in 10 min 30 % B to 60 % B in 5 min Column wash 60 % B to 10 % B in 1 min UV detector variable wavelength detector 204 nm for 4.5 min 368 nm for 1.5 min 204 nm for 4 min 254 nm for 5 min 204 nm, standard cell Column compartment temperature 40 C Stop time 16 min Post time 5 min Injection volume 5 l

15

Sulfamethoxazole

10 Wavelength-switching 5 275/343 nm 250/343 nm 0 250/503 nm 0 2 4 6 8 10 12 14 16 18 Multiwavelength detection

Time [min]

Figure 4 Analysis of trimethoprim and sulfamethoxazole using a fluorescence detector (column: Zorbax SB-C18, 5 m, 50 x 2.1 mm)

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Table 3 shows the performance of the HPLC method for the six antibacterial drugs shown in figure 3.
Compound LOD for S/N=2 (mg/l)* 0.1 0.1 0.1 0.1 0.1 0.1 Precision of RT (RSD of 10 runs) (100 mg/l)* 0.06 0.03 0.07 0.04 0.04 0.02 Precision of Area (RSD of 10 runs) (100 mg/l)* 0.18 0.25 0.17 0.20 0.17 0.17 Linearity (Cor. factor) (1-100 mg/l)* 1.00000 1.00000 1.00000 1.00000 1.00000 0.99990

Equipment
Agilent 1100 Series Quaternary pump (includes vacuum degasser) Thermostatted autosampler Thermostatted column compartment Variable wavelength detector, standard flow cell, 10-mm path length, 13-l cell volume

Hydroxybenzotriazole Chloramphenicol Trimethoprim Sulfamethoxazole Furazolidone Nalidixic acid

* Injection volume: 5 l

Table 3 HPLC method performance for the six antibacterial drugs shown in figure 3 The methods presented in this Application Brief show easy but reliable and precise analyses of the antibacterial drugs amoxicillin, ampicillin, penicillin G, penicillin V, minocycline, tetracycline, doxycycline, nalidixic acid, trimethoprim, sulfamethoxazole, chloramphenicol, hydroxybenzotriazole and furazolidone. The values for LOD, precision of RT, precision of area and linearity show the good performance of the analyses.

Alternative: Vacuum degasser Binary pump Diode-array detector, standard flow cell 10-mm path length, 13-l cell volume Fluorescence detector, standard flow cell, 8-l cell volume Agilent ChemStation + 3D software
Columns Zorbax SB-C18, 3.5 m, 4.6 x 75 mm (Agilent part number 866953-902) Recommended: Guard cartridges Zorbax SB-C18, 5 m, 4.6 x 12.5 mm (Agilent part number 820950-920, 4/pk)
Udo Huber is application chemist at Agilent Technologies, Waldbronn, Germany. For more information on our products and services, visit our worldwide website at http://www.agilent.com/chem
Copyright 1998 Agilent Technologies Released 08/98 Publication Number 5968-1078E

Note: Since the methods were specifically developed on the Agilent 1100 Series system you might not be able to reproduce them on an older system or even on a new system with lower performance. To avoid sample decomposition a cooled autosampler, for example, the Agilent 1100 Series thermostatted autosampler is required. A programmable variable wavelength detector, for example, the Agilent 1100 Series variable wavelength detector is required because of the different absorbance maxima of the compounds. A fluorescence detector, for example, the Agilent 1100 Series fluorescence detector can also be used.

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