中药中理化常数检测方案

2 Rapid Screening Method for VeterinaryAntibiotics Using an Advanced Solid CoreUHPLC Column and UHPLC System withMS/MS Detection Gordon Wilson and Derek Hillbeck， Thermo Fisher Scientific， Runcorn， UK Key Words Vanquish， Accucore， antibiotics， sulfonamides， penicillins，macrolides，screening Abstract This application shows the advantages of using the Thermo ScientificTMAccucoreTM VanquishTM C18 UHPLC 1.5 pm column and Vanquish UHPLCsystem for the analysis of thirty-six antibiotics. Advanced capabilities of the Vanquish UHPLC system allow the AccucoreVanquish UHPLC columns to be operated at high flow rates that enabledevelopment of rapid analytical methods while maintaining performance.The need for chromatographic separation of isobaric compounds prior toMS detection is also highlighted. The Accucore Vanquish UHPLC column and VanquishUHPLC system were designed in combination to achievethe best possible chromatographic performance. Thesystem is optimized to reduce extra column banddispersion and allow users to significantly improve theseparation power in their analytical assays. By exploitingthe 1500 bar high pressure capability of the VanquishUHPLC system， flow rates can be increased whilemaintaining peak capacity， resulting in shorter methodtimes and increased assay throughput. Experimental Details Consumables Part Number Accucore Vanquish C18， 1.5 pm UHPLC column， 100×2.1 mm 17101-102130 LC-MS grade 18 MA water from Thermo Scientific Smart2PureTM system 50129845 Fisher ChemicalTM LC-MS grade methanol 10653963 Fisher Chemical analytical grade formic acid 10559570 Thermo ScientificTM VirtuosoTM 9 mm wide opening， 2 mL screw thread vial and cap kit 60180-VT400 Solutions of the 36 compounds shown were prepared by dissolving 1 mg amounts in 1 mL of water/methanol (1：1 v/v)to produce 1 mg/mL primary solutions. Where required， the diluents proportions were altered to reflect the solubility of thematerials and ensure sample solubility. Serial dilutions were made with water/methanol (90：10 v/v) to produce a100 ng/mL working solution. Vial labeling was supported by the Virtuoso Vial Identification System. Instrumentation Part Number Vanquish UHPLC system consisting of： Binary pump H VH-P10-A Split sampler HT VH-A10-A Column compartment H VH-C10-A Thermo ScientificTM TSQ VantageTM triple quadrupole mass spectrometer Virtuoso Vial Identification System 60180-VT-100 LC/MS Conditions UHPLC column： Accucore Vanquish C18， 1.5 pm， 100×2.1 mm Mobile phase A： 0.1% formic acid in water Mobile phase B： 0.1% formic acid in methanol Flow rate： 400 pL/min Column temperature： (Forced air mode) with active eluent preheating set to 40 ℃ Injection details： 2pL Gradient： Refer to Table 1 Time (min) %B 0.000 10 4.375 90 5.000 90 5.125 10 8.750 10 Table 1： LC gradient conditions Compound detection was achieved by selected-reaction monitoring (SRM) experiments on a TSQ Vantage triplequadrupole mass spectrometer.Source and tuning conditions are set out in Table 2 and compound specific parameters for the 36 different SRMtransitions are shown in Table 3， together with the analyte retention time when using a 400 uL/min flow rate. Data Processing Software： Thermo ScientificTM XcaliburTM 2.1 (MS control) Thermo ScientificTM DionexTM ChromeleonTM 7.2 Chromatography Data System Parameter Setting lonization conditions HESI Polarity Positive Spray voltage (V) 5000 Vaporizer temperature (C) 500 Sheath gas pressure (Arb) 75 Aux gas pressure (Arb) 20 Capillary temperature (℃) 380 Collision pressure (mTorr) 1.5 Scan time (s) 0.005 Q1 resolution (FWHM) 0.7 Q3 resolution (FWHM) 0.7 Table 2： TSQ Vantage MS/MS experiment conditions Table 3： Compound SRM transition details and retention time Results By exploiting the high pressure capabilities of the Vanquish UHPLC system in conjunction with theAccucore Vanquish UHPLC column and a simple binary gradient， it was demonstrated that ascreening method for 36 compounds within a 5 minute detection window (and a full method cycletime of less than 9 minutes) can be achieved (Figures 1-4). Using a 400 pL/min flow rate the system pressure at the start of the gradient was 840 bar， rising toa maximum of 1210 bar during the gradient cycle. The Vanquish UHPLC system is able toroutinely operate at these pressure conditions. Time (min) Figure 1： Overlaid selected-reaction monitoring chromatograms showing detection of 36 compounds within a5 minute detection window Time (min) Figure 2： Selected-reaction monitoring chromatogram for sulfamethoxazole， representative of a sulfonamideantibiotic Figure 3： Selected-reaction monitoring chromatogram for erythromycin， representative of a macrolideantibiotic Figure 4： Selected-reaction monitoring chromatogram for nafcillin， representative of a penicillin antibiotic When isobaric compounds are present in the screening portfolio it is not possible to rely solely onthe mass resolution capability of the mass spectrometer，particularly as structurally similarcompounds can have common precursor and product ions. In these cases the separation of thecomponents using chromatography provides a clear advantage. The compounds of interest here，sulfadoxine and sulfadimethoxine， are structural isomers with the same molecular weight， productand precursor ions but slightly different structures. In this situation multiple peaks were observed but were able to be individually resolved (Figure 5).Without this chromatographic resolution the identification of these compounds in a screening setwould be compromised. Figure 5： Selected-reaction monitoring chromatogram showing the chromatographic separation of structural.isomers Conclusion This application note demonstrates the advantages of using the Accucore Vanquish C18 1.5 umUHPLC column and Vanquish UHPLC system. This solution： Delivers a rapid screening UHPLC-MS method for 36 antibiotics Delivers a method time less than five minutes in duration Exploits the high pressure capabilities of the Vanquish UHPLC system Supports separation of isobaric compounds prior to MS detection For research use only thermoscientific.com/accucore C2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information ispresented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that mightinfringe the intellectual property rights of others. Specifications， terms and pricing are subject to change. Not all products are available in all countries.Please consult your local sales representative for details. 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