focus on Chromatography


Monolithic Silica Columns for Simple and Fast LC-MS Analysis of Antibiotics in Mammalian Tissue and Body Fluid and in Pharmaceutical Formulations


Stephan Altmaier, Merck Millipore, Frankfurter Str. 250, 64293 Darmstadt, Germany


This work describes fast and straightforward high performance liquid chromatography methods with UV or mass spectrometry detection for the analysis of antibiotics in mammalian tissue or urine and in pharmaceutical formulations. The monolithic and very robust silica structure of the columns used in this work allows for short sample preparation procedures. All samples were separated on C18 reversed phase columns via gradient elution profi les and directly transferred to UV or MS for the analysis of all components. This setup enabled the identifi cation of antibiotics in samples such as urine or liver within very short analysis times and with a sample preparation step kept as short as possible.


Antibiotics are natural products of metabolism of bacteria, fungi or organisms such as plants and amphibians, generated in order to fi ght an infection or to gain advantage in genetic selection. Examples for such natural antibiotics are allicin from garlic, Echinacea plant extract, honey containing antimicrobial enzymes or cinnamon.


From a more medical point of view, antibiotics are a class of drug substances blocking metabolic processes of microorganisms. As a result, reproduction and viability of these microorganisms is prevented. Antibiotics are utilised in the treatment of a vast number of infections caused by bacteria. Depending on their activities, they can be referred to as bacteriostatic (stopping bacteria from reproducing, but not crucifying them) or bactericide (stopping reproducing and crucifying). The most important classes of antibiotics are penicillin, cephalosporins, carbapenems, fl uoroquinolones, macrolides, aminoglycosides and tetracyclines, differing in their effectiveness towards various types of bacteria. Antibiotics used for therapy are nowadays produced fully or partially synthetic or via biotechnological processes.


One drawback of antibiotics is their (ab)use in intensive mass animal farming. Here, these drugs are often applied in a preventive manner against various illnesses and in order to improve the results in animal fattening. As a result antibiotic resistant bacteria are selected. These and antibiotic compounds are then released into the environment via manure spreading, and the biological activity of antibiotics in ground water and soil further increases the number of resistant bacteria. With respect to humans, the unspecifi c or unnecessary prescription and taking of antibiotics also leads to the development of bacterial resistance. In addition, the excretion of excess antibiotics via urine or faeces leads to the same problems as described above for animal farming. Such approaches in both human and veterinary medicine clearly foil antibiotic stewardship, i.e., a responsible case-to-case indication utilising antibiotics.


As a consequence of their lax handling, antibiotics are nowadays omnipresent in the environment and in the food chain, namely in meat and dairy products, but also in vegetables. This makes a reliable, fast and robust analysis of antibiotics in various complex matrices necessary. In this work several straightforward methods combining reversed phase liquid chromatography (LC) separation and ultraviolet (UV) and mass spectrometry (MS) detection are presented for the analysis of four different antibiotics in food and urine (Figure 1). Robust monolithic silica column technology was utilised, enabling fast chromatographic runs and the detection of all analytes without the need for complex sample preparation procedures and costly UPLC systems.


1 2


Table I: List of antibiotics analysed in this work: Names, corresponding monoisotopic mass, relevant MS peaks (calculated) and molecular ion formulas.


Antibiotic compound


Cephalosporin C Cefaclor Cefalotin


Phenoxymethylpenicillin


Monoisotopic mass / g/Mol


415.1 367.0 396.0 350.1


Relevant MS peak / m/z


416.1 368.0 337.0


351.1, 160.0


Experimental Materials and Methods


The HPLC system used was a Dionex Ultimate 3000 (Thermo Scientifi c Dionex Corporation, Sunnyvale, California, USA) equipped with a UV detector (detection wavelength 254 nm), a Chromolith®


FastGradient RP-18 endcapped 50-2 mm analytical monolithic silica column and a Chromolith®


RP-18 endcapped 5-2 mm guard cartridge (both Merck Millipore, Darmstadt, Germany). The data acquisition was performed with Chromeleon software.


A Bruker Esquire 6000plus mass spectrometer with an ion trap and an on-line electrospray ionisation (ESI) source operated in positive mode was utilised in the m/z range scan from 100-500. Flow and temperature of the dry gas was set to 12 L/min and 365°C, respectively, nebuliser gas pressure was 2.8*105 Pa. Trap conditions: Max. accu time 100 ms, target 200.000, averages: 2.


Sample Preparation Bovine liver was purchased at a local butcher. Detailed information about sample preparation or fortifi cation can be found in the different sections below.


Antibiotics Stock Solution Stock solutions were prepared by ultra wave supported dissolution of three antibiotics in 10 mL water and a fi ltration of the resulting solutions using a Millex®


syringe fi lter driven 3


unit PTFE 0.45 µm. The fi nal concentrations of the single compounds in each solution were: Cephalosporin C (CPS) 217 mg/L, cefaclor (CFC) 2343 mg/L, cefalotin (CPT) 158 mg/L. The fi nal stock of all three antibiotics (‘CCC’) was then prepared by combining 9.52, 198.1 and 652.4 µL of the three antibiotic stock solutions of CFC, CPT and CPS.


4 Antibiotic Formulation


Figure 1. Chemical structures of antibiotics analysed in this work. 1. Cephalosporin c, 2. Cefaclor, 3. Cefalotin, 4. Phenoxymethylpenicillin.


An antibiotic formulation containing phenoxymethylpenicillin (penicillin V) and various preservatives (sodiummethyl-4-hydroxybenzoate, sodiumpropyl-4-hydroxybenzoate, sorbic acid) and additives (saccharose, sodium cyclamate, saccharin, citric acid, carmine (E120)) was utilised to spike human urine. The stock solution was prepared by dissolving 34.9mg of the dry powder in 50 mL water and fi ltering of the resulting solution using a Millex® syringe fi lter (0.45 µm pore diameter). The concentration of phenoxymethylpenicillin in the fi nal solution was 69.8 mg/L.


INTERNATIONAL LABMATE - APRIL 2014


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84