Introduction The Ostro sample preparation plate enables simple, rapid LC-MS determination of basic drugs and related compounds in biological samples. The Ostro sample plate facilitates liquid-liquid extraction, protein precipitation, filtration, and subsequent removal of phospholipids from the final extract. In this paper, the Ostro sample plate was used to screen the veterinary drug residues in milk. The use of this sample plate can effectively precipitate proteins and remove potentially interfering substances in a few simple steps to complete the preparation of the milk sample. As the sample is passed through the sample plate, the protein is removed by filtration and the phospholipid interferent is removed by being retained on the patented adsorbent. After completing the simple sample preparation step, the reconstituted sample was analyzed using UPLC-MS/MS. To demonstrate the applicability of this method, we have selected representative compounds from the main veterinary drug classes, including tetracycline, fluoroquinolone, sulfonamides, macrolides, beta-lactams, and beta-adrenergic drugs. Sample Preparation Transfer 125 μL of milk to the well of the sample plate (sample volume must not exceed 125 μL). Add 375 μL of 0.2% formic acid in acetonitrile (ACN). Pump a few times to mix them thoroughly. Elution to the collection plate. Add 100 μL of 200 mM ammonium formate in methanol/ACN (50:50) and mix well. Evaporate dry and re-dissolve in 100 μL of ACN/25 mM aqueous ammonium formate (25:75). Figure 1 shows a typical SPE setup when using an Ostro sample plate. Results and discussion Multi-residue analysis The results for the Ostro sample plate were comparable to the previously published method 1 . However, Ostro sample plates operate less time and are more suitable for high throughput analysis. Using this method, a typical analyst can prepare 10 or more samples in a few minutes, while the previous method takes several hours. Figure 2 shows a typical LC-MS chromatogram of enrofloxacin (10 μg/L) matrix-matched standards. Although most of the other compounds showed similar analytical performance when analyzed using this method, quantitative analysis of chlortetracycline, oxytetracycline and lincomycin at concentrations of 10 μg/L was not possible. An alternative method for tetracycline has recently been developed 2 . Table 2 shows the recovery data for the observed multi-residue milk analysis. Phospholipid removal Milk contains large amounts of phospholipids, representing approximately 1% of the total fat content of 3. In LC separation, these phospholipids directly interfere with the post-eluting compound. Furthermore, the results indicate that the accumulation of phospholipid residues deteriorates the performance of reversed-phase LC separation 4 . Figure 3 shows a UPLC-MS/MS chromatogram of a milk sample analyzed using an MRM channel (m/z 184 > 184) selected to identify phospholipids. The figure below shows the results obtained using a Ostro sample plate to process milk samples. The top panel shows the results obtained with protein precipitation (not purified by SPE). The Ostro sample plate is effective in removing more than 98% of phospholipids. in conclusion ■Demonstrates the rapid extraction of multiple veterinary drugs from milk using the Ostro sample preparation plate. references 1. Optimized Extraction and Cleanup Protocols for LC-MS/MS Multi-Residue Determination of Veterinary Drugs in Edible Muscle Tissues, Waters Application Note 720004144en 2. HPLC/UV Determination of Tetracyclines in Milk Using Mixed-Mode SPE and eXtended 3. Contarini, G and Povolo, M., Phospholipids in Milk Fat: Composition, Biological and Technological Significance, and Analytical Strategies, Int. J. Mol. Sci, 14(2), 2808–2831 (2013) 4. Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method, Waters Application Note 720004046en
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â– In addition to tetracycline, this method shows good screening performance for all other types of tested compounds.
â– Use Ostro straight-through sample preparation plates to effectively remove almost all phospholipids from milk extracts.
Performance [XP] 2.5 μm Columns, Waters Application Note 720004582en
Screening of multiple residues of veterinary drugs in milk using Ostro sample preparation plates in combination with LC-MS/MS>
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