13 Urine Spiked with Antibiotics
Human urine was first centrifuged at 4500 rpm for 45 minutes and filtered utilising a Millex®
syringe filter (0.45 µm pore diameter). Urine was then combined with the antibiotic formulation in a 1:1 volume ratio.
Bovine Liver Spiked with Antibiotics One blank and one bovine liver sample spiked with the CCC mixture was prepared. 0.98 g of liver were broken up and mixed with 0.6 mL of CCC solution. In parallel, a blank sample (1.02 g of liver) was prepared in a similar manner but without the addition of antibiotics. After one hour both samples were combined with 10 mL of ACN/water 80:20 (v:v), homogenised using an Ultra-Turrax T25 (IKA) and centrifuged at 4500 rpm for 15 minutes. The clear and yellowish supernatant was decanted off into two glass tubes and ACN was evaporated during 60 minutes at a gas flow of 1 bar and a temperature of 40°C with a TurboVap instrument (Biotage). The residue was transferred to a LiChrolut®
RP-18e 500 mg cartridge
preconditioned with 5 mL of ACN and two times 5 mL water. The yellow band was eluted into a centrifuge tube with 5 mL ACN and residual eluent was vacuumed. 1 mL water was added to the solution and after shaking for 30 seconds the mixture was again concentrated with a TurboVap (120 minutes, 40°C, 1 bar compressed air). The samples were then filtered via a Millex® HPLC vials.
syringe filter (0.45 µm pore diameter) and transferred to Results and Discussion
A prerequisite in modern LC-MS analysis is high sample throughput, or in other words: A fast LC-MS method in order to avoid a bottleneck in the system sample preparation- separation/detection-analysis. Hence, a mixture of the three antibiotics cephalosporin, cefaclor and Cefalotin was prepared and a fast gradient LC method was developed in order to achieve baseline separation of all analytes. Detection was performed by both UV and MS and a monolithic silica column was chosen as a stationary phase to allow for a low backpressure separation at a comparably high flow rate (Figure 2). All compounds eluted within less than two minutes and a baseline separation of the narrow peaks was achieved.
Figure 3. LC-MS chromatograms of a bovine liver blank run (top) and of a standard solution of three antibiotics spiked in bovine liver (bottom) separated on Chromolith®
FastGradient®
endcapped 50-2 mm analytical monolithic silica column and Chromolith®
RP-18 RP-18
endcapped 5-2 mm monolithic silica guard column. For conditions and peak annotiations see Figure 2.
The third example simulates excreted human urine after antibiotic medication. Urine pretreatment followed by spiking of the resulting liquid with an antibiotic syrup formulation. This sample was analysed via LC-MS utilising a combination of monolithic silica analytical and guard columns and a short two-step gradient (Figure 4). For the blank syrup no guard column was applied. The chromatogram of the formulation reveals only four signals, of which the first can be attributed to sugar eluting in the void volume. The intensive and narrow peak at approximately 4.3 minutes is caused by phenoxymethylpenicillin, while two additional signals at 3.1 minutes remain unassigned. The spiked urine sample shows several peaks equally distributed throughout the whole chromatographic runtime. Phenoxymethylpenicillin is eluting as a narrow and almost baseline separated peak at 4.9 minutes and was identified by its spectrum pattern (m/z 160.0 and 351.1, see inset in Figure 4 bottom). A visible shift in retention of this signal (in comparison to the blank syrup sample) is caused by the use of a guard column.
Figure 2. LC-MS chromatogram of a mixture of a standard solution of three antibiotics separated on Chromolith®
FastGradient® RP-18 endcapped 50-2 mm analytical monolithic silica column.
Detection: UV (254 nm, left) and positive ESI-MS (m/z 100 – 500), base peak chromatogram (BPC, right). Mobile phase A: acetonitrile + 0.1% formic acid, mobile phase B: water + 0.1% formic acid; flow rate 0.4 mL/min; gradient: 0’ 5% A, 2’ 95% A, 5’ 95% A; pressure drop 23 bar. Sample: 1 Cephalosporin c, 2 Cefaclor, 3 Cefalotin.
In a second example the same mixture of antibiotics was utilised to spike homogenised bovine liver. After spiking and centrifugation a typical solid phase extraction (SPE) protocol was performed. Undissolved particulate matter as well as salts were removed applying a pre-packed SPE cartridge containing reversed phase stationary phase material. LC-MS analysis of the resulting extract as well as of an unspiked blank control sample was achieved utilising the same gradient as applied in Figure 2. A 50-2 mm reversed phase monolithic silica column was used in these runs. In order to protect the analytical column a 5-2 mm RP-18e guard column was additionally installed. MS data of the analysis of the blank sample displays a bunch of matrix peaks in the range of 0.5 – 3 minutes, with several unassigned polar compounds eluting mainly in the void volume and possibly interfering with the cephalorsporin signal (Figure 3 top). Hence, the gradient run provides a trade-off between speed of analysis and resolution. The analysis of the spiked bovine liver extract confirmed some peak overlay in the < 1 min region, nevertheless all antibiotics were identified via MS detection (Figure 3 bottom). Later eluting analytes cefaclor and Cefalotin were detected as baseline separated peaks. No or only small peaks are eluting in the range of 3-5 minutes, revealing the excellent suitability of the applied sample preparation procedure in the analysis of antibiotic contamination in mammalian tissue.
Figure 4. LC-MS chromatogram of an antibiotic drug formulation (top) and of the same formulation spiked in human urine (bottom). Insert: MS spectrum of 1 (phenoxymethylpenicillin) at 4.91 min. Sample analysis was performed on Chromolith®
FastGradient®
50-2 mm analytical monolithic silica column coupled with a Chromolith®
RP-18 endcapped RP-18
endcapped 5-2 mm monolithic silica guard column. Detection: Positive ESI-MS (m/z 100 – 500), base peak chromatogram (BPC). Mobile phase A: acetonitrile + 0.1% formic acid, mobile phase B: water + 0.1% formic acid; flow rate 0.4 mL/min; gradient: 0’ 5% A, 3.5’ 70% A, 5’ 95% A, 7’ 95% A.
Conclusion
This work shows that monolithic silica technology combined with fast sample preparation methods is a robust system ideally suited for the analysis of antibiotics in complex food and urine matrices. Particulate matter and highly unpolar matrix components were removed via the sample preparation process, and most of the more polar compounds were separated from the antibiotics applying fast gradient runs. The target analytes were then easily identified via UV or MS detection. Due to the low backpressure characteristics of the monolithic silica columns it was possible to perform all chromatographic runs on standard HPLC systems. In order to protect the analytical column and to increase it’s lifetime, a guard column was utilised.
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