Automated analysis of macrolide antibiotics in milk

Claude R. Mallet, Dimple D. Shah
Waters Corporation, Milford, MA, US

Introduction

In 1938, the US government passed the Federal Food, Drug, and Cosmetic Act (FFDCA, FDCA, or FDA), which gave the US Food and Drug Administration (FDA) the power to inspect food, medicine, and cosmetics. Today, the FDA manages all domestic and imported foods other than meat, poultry, and certain dairy products managed by the US Department of Agriculture. Now, all milk is worked out by the FDA, the US government, and milk producers to monitor drug residues. In the 2007 Financial Yearbook 1, more than 4 million samples were analyzed for a wide range of antibiotics, such as lactams, macrolides and tetracyclines. For example, the US FDA recommends a maximum residue (MRL) of 50 mg/kg for a macrolide antibiotic tilmicosin in milk, which is also accepted by the European Union. Many countries follow the US Food and Agriculture Organization/World Health Organization (WHO) maximum residue of 40 μg/kg instead of setting the maximum residue for each drug.

Macrolides are a group of drugs whose activity is derived from the presence of a macrolide ring, while the macrolide ring may have one or more deoxy sugars, often di-deoxymethyl hexose and deoxygenated Glycosamine. Lactones are often 14-, 15- or 16-ringed. Macrolide antibiotics are widely used in veterinary medicine to treat many diseases, such as respiratory diseases in lactating dairy cows, mastitis with no clinical symptoms, or they can be used as feed additives to promote growth. If the milk extraction period is too short, antibiotic residues may still be present, which can cause direct toxic side effects (allergic reactions) to consumers 2 .

Figure 1 Macrolide antibiotics

Offline SPE extraction protocol

Traditionally, microbiological test 3 has been used to monitor antibiotics in food matrices. These programs have very good quantitative results, but are increasingly lacking in high specificity. With the widespread use of modern laboratory LC/MS or LC/MS/MS systems, the multi-residue method is a more accepted method of routine work.

Analytical protocols that include food matrices are often complex and require hours of manual manipulation. For example, a drinking water regimen (lower sample complexity) consists of the following steps: direct loading to the SPE cartridge, small wash steps, elution, and finally evaporation and reconstitution with the mobile phase. Solid matrices such as food samples (higher sample complexity) must be first homogenized with aqueous or organic extraction buffers, followed by some centrifugation steps, liquid-liquid extraction (optional), and finally before implementing the SPE extraction protocol. Dilute with aqueous solution. For milk analysis, the most common extraction protocol is to precipitate protein 4 with acetonitrile. The mixture is then centrifuged for a few minutes. The supernatant can be further purified by other extraction techniques.

Offline SPE milk solution

Step 1: Add 5 mL of milk to a 50-mL centrifuge tube. Step 2: Add 15 mL of acetonitrile. Step 3: Vortex and centrifuge at 3200 RPM for 15 min.
Step 4: Transfer the supernatant to a 50-mL centrifuge tube. Step 5: Add 20 mL of n-hexane. Step 6: Vortex for 15 min.
Step 7: Centrifuge at 3200 RPM for 15 min
Step 8: Remove the n-hexane layer Step 9: Evaporate the acetonitrile/milk supernatant to 3 mL
Step 10: Add 15 mL of 0.1 M, pH 8.0 Sulfate Buffer Step 11: Activate Oasis® HLB (6cc) with 10 mL of Methanol
Step 12: Activate Oasis® HLB (6cc) with 10mL of water
Step 13: Activate Oasis® HLB (6cc) with 10mL 2% NaCl
Step 14: Activate Oasis® HLB (6cc) with 2mL 0.1M phosphate with a pH of 8.0
Step 15: Upload the reconstituted sample (Step 10)
Step 16: Wash with 5 mL of water Step 17: Wash with 5 mL of 40% aqueous methanol. Step 18: Allow Oasis HLB to drain for 5 min.
Step 19: Elute with 5 mL of 95% aqueous methanol. Step 20: Evaporate near dry (45 min)
Step 21: Reconstitute with 1 mL of the initial mobile phase. Step 22: Filter the extract with 0.45 μm of polyvinylidene fluoride (PVDF)

Online SPE extraction protocol

Previous publications (online SPE/LC/MS/MS Parts I, II and III) introduced a novel automated extraction/analysis technique: the Waters AquaAnalysis system. The overall solution platform offers several benefits: reduced manual handling, reduced solvent consumption and reduced sample volume. The time-consuming evaporation/reconstitution step is eliminated by using the online SPE/LC/MS/MS technology platform.

Online SPE/LC/MS/MS technology is not only suitable for drinking water analysis. Drinking water samples can be loaded directly into the autosampler, followed by a fully automated computer-controlled extraction/analysis step. For food analysis, solid samples are first converted to liquid form. The analysis begins with homogenization to release the bound analyte to the liquid phase extraction buffer. Homogenization requires a centrifugation step and the supernatant is then removed. At this point, the analyte of interest is present in a suitable form to facilitate other purification, concentration or extraction steps, such as liquid-liquid extraction (LLE) and SPE. The food sample extraction/analysis process now requires only homogenization, centrifugation and dilution steps by using an online SPE protocol under software control.

Online SPE milk program

Step 1: Add 1 mL of milk to a 50 mL centrifuge tube. Step 2: Add 20 ul of ammonia in a certain concentration. Step 3: Add 2 ml of acetonitrile (2:1 ratio)
Step 4: Vortex and centrifuge for 15 min at 3500 RPM
Step 5: Transfer 2 mL of the supernatant to a 20 ml sample vial Step 6: Add 18 mL of water Step 7: Inject 5 mL

experiment

The MRM conditions used for macrolides are listed in Table 1. The SPE extraction column used was Waters® Oasia HLB 2.1 x 20 mm, 25 μm, neutral pH loading, mobile phase (no additives). The analytical column used was XBridgeTM C18 2.1 x 50 mm 3.5 μm, low pH mobile phase elution. Cleaning and reactivation parameters are listed below.

SPE and LC working conditions

Load pump: Line A: 100% water line B: 100% methanol sample flow rate: 4.0 mL/min
Cleaning solution: 20% methanol / acetonitrile ( 30/70 ) +0.5% formic acid rebalancing pump: A : 50/50 methanol / acetone
B : 80/20 ethyl acetate / acetone
C : 80/20 n-hexane/acetone rebalancing flow rate: 4.0 mL/min
Extraction column: Oasis HLB 2.1x 2 mm , 25.0 μ m
Analytical column: XBridge C18 2.1x 50 mm , 3.5 μ m

Results and discussion

Figure 2 is a representative extracted ion chromatogram of a milk sample with 0.5 ppb of each antibiotic added. Antibiotics have good signal and peak shape at trace levels. For food samples, the complexity of the sample is higher than that of the drinking water sample, and the cleaning step needs to be optimized to maximize the removal of interfering substances without eluting the analyte of interest. Figure 3 shows a chromatogram of 100 ppb erythromycin added with 5%, 10%, 20%, 30%, and 40% methanol. In Figure 2, a 20% methanol wash produces a higher signal than a 5% methanol wash. The weak signal of 5% methanol can be attributed to the ion suppression effect. However, when the organic ratio in the washing step is increased too much, the sensitivity of the signal tends to decrease due to partial elution.

Figure 4 shows the calibration curve for erythromycin concentration from 1.0 ppb to 500.0 ppb with a linear coefficient of 0.9902. Table 2 compares the online and offline SPE extraction schemes side by side, from literature to processing natural milk samples to ideal forms for LC/MS or GC/MS analysis. When evaluating online SPE/LC/MS/MS and off-line solutions, the usual extraction procedures and the overall application of solvents, laboratory personnel and time are important. Other food samples have a variety of configurations and may require additional processing prior to SPE extraction. For example, milk samples require simple protein precipitation and centrifugation before further purification with SPE devices. For like

Figure 2 shows the extracted ion chromatogram of 0.5 ppb milk.

Figure 3 Optimized cleaning of milk extraction

Figure 4 erythromycin calibration curve

Solid samples such as fruits and vegetables, low temperature milling or homogenization steps using extraction buffers are a preferred method. For the usual extraction procedures, it is easy to find literature reports that use a wide range of volumes and times for each extraction step. Typically, using the SPE protocol, the loading, washing and elution volumes are all based on the bed size of the SPE unit. This can first ensure the overall wetting of the SP E adsorbent in each step. Once this value is set, this can be multiplied several times (depending on the compound) to ensure multiple volume displacements to minimize the cleaning step, clear the interference and elute steps to maximize recovery. In actual operation, an increase in sample trapping capacity on the column bed requires an increase in volume in the SPE procedure; this ultimately increases sample preparation time and solvent consumption.

in conclusion

In the past few years, many countries have made great efforts to further improve the safety of their domestic and imported food supplies so that consumers can enjoy a safe food supply. Because of this, many analytical methods are available and used in routine analysis. Increased solvent usage and increased demand for laboratory personnel

Plus, the use of automation platforms will definitely meet these requirements. This application note describes the automated extraction and analysis of antibiotics in milk samples. Traditional pre-extraction steps are extremely time consuming; they require a greater amount of chemicals and a wide variety of reagents. The time to process milk samples using the Waters AquaAnalysis system is approximately 20 minutes, which can be reduced by 70-90% compared to the off-line solution.

Table 2 Solvent consumption and processing time for offline and online extraction protocols for milk samples

references

[1] US FDA National Milk Drug Residue Database. Food and Drug Administration, Center for Food Safety and Drug Administration, 2007.
Available online: http://~ear/p-mis.html
[2] WA Moats, MB Medina. Veterinary Drug Residues, ACS Symposium Series 636, American Chemical Society, Washington, DC, 1996, p.5.
[3] 43rd Meeting of the Joint FAO/WHO Expert Committee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food, WHO Tech. Rep Ser., No 855, 1995, p.85.
[4] J Wang, D Leung, SP Lenz. J. Agric. Food Chem. 54: 2873, 2006.|
[5] M Dubois, D Fluchard, E Sior, PH Delahaut. J. Chromatogr. B.753: 189, 2001.
[6] SB Turnipseed, WC Andersen, CM Karbiwnyk, MR Madson, KE Miller. Rapid Commun. Mass Spectrom. 22: 1467, 2008.

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