中药中理化常数检测方案

智能文字提取功能测试中

Derek Hillbeck， Thermo Fisher Scientific， Runcorn， UK Key Words Vanquish， Accucore， opiates， benzodiazepines， amphetamines， drugs ofabuse， forensic， toxicology Abstract This application shows the advantages of using the Thermo ScientificTMAccucoreTM VanquishTM C18 UHPLC， 1.5 um column and Vanquish UHPLCsystem for the analysis of 47 illicit drugs. 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. Introduction There is increasing demand to provide rapid and selectivescreening techniques for an expanding range of illicitdrugs for forensic methodology and workplace drugscreening. Creating screening methods for multipleanalytes is more cost-effective than dedicated methods forfewer analytes. Reduced analysis times provide forquicker release of data， reduced costs per assay， andgreater sample throughput overall. Other methodologies exploit the high mass resolutioncapabilities of specialty mass spectrometers[1].Wherethese are not readily available， and where commonprecursor and product ions are present， there is arequirement for good chromatographic resolution， priorto MS detection. Accucore Vanquish UHPLC columns use Core Enhanced TechnologyTM to facilitate fast and highly efficientseparations. This next-generation column features 1.5 umsolid core particles that are not totally porous， but insteadhave a solid core and a porous outer layer. The optimizedphase bonding creates a high coverage， robust phase. Thiscoverage results in a significant reduction in secondaryinteractions and delivers highly efficient peaks. The tightlycontrolled 1.5 um diameter of Accucore Vanquishparticles， in combination with controlled manufacturingprocesses，results in a column that delivers the increasedchromatographic performance required for rapidscreening methods commonly used in forensic toxicology. 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， the 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 MQ water from Thermo ScientificTM Smart2PureTM system 50129845 Fisher ChemicalTM LC-MS grade methanol 10653963 Fisher Chemical analytical grade formic acid 10559570 Fisher Chemical ammonium acetate 10598410 Thermo ScientificTM VirtuosoTM 9 mm wide opening， 2 mL screw thread vial and cap kit 60180-VT400 Sample Preparation Solutions of the 47 compounds shown in Table 3 were prepared by dissolving 1 mg amounts in 1 mL of water/methanol(1：1v/v) to produce 1 mg/mL primary solutions. Where required， the methanol proportion was increased to ensure samplesolubility. Serial dilutions were made with water/methanol (90：10 v/v)to produce 100 ng/mL working solutions. Vial labeling is 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 um， 100×2.1 mm Mobile phase A： 10 mM ammonium acetate in water Mobile phase B： 0.1% formic acid in methanol Flow rate： 500 pL/min Column temperature： (Forced air mode) with active eluent preheating set to 50 ℃ Injection details： 2pL Gradient： Refer to Table 1 Time (min) %B 0.00 10 0.16 10 2.88 90 3.20 90 3.28 10 5.60 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 47 different SRMtransitions are shown in Table 3， together with the analyte retention time. 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) 4500 Vaporizer temperature (C) 500 Sheath gas pressure (Arb) 75 Aux gas pressure (Arb) 20 Capillary temperature(C) 380 Collision pressure (mTorr) 1.0 Scan time(s) 0.005 Q1 resolution (FWHM) 0.7 Q3 resolution (FWHM) 0.7 Table 2： TSQVantage MS/MS experiment conditions Table 3： Compound SRM transition details and retention time Standard Precursor lon S-Lens (0) Product lon Collision Energy (M) RetentionTime (min) Norbuprenorphine 414 160 187 36 2.39 PCP(phencyclidine) 244 53 91 34 2.43 1，5-dimethyl-3，3-diphenylpyrrolodone (EDDP) 278 120 234 29 2.46 Clonazepam 316 95 270 23 2.66 Tilidine 274 76 155 18 2.77 0xazepam 287 87 241 20 2.81 Methadone 310 84 265 13 2.81 Lorazepam 321 100 275 20 2.82 Alprazolam 309 101 281 24 2.84 Flurazepam 388 100 315 21 2.95 Fentanyl 337 106 188 22 2.97 Nordiazepam 271 100 140 27 2.99 11-nor-A-9-carboxy-THC 345 104 193 24 3.49 Buprenorphine 468 138 187 39 3.75 Table 3 (continued)：Compound SRM transition details and retention time Results By exploiting the high pressure capabilities of the Vanquish UHPLC system， in conjunction withthe Accucore Vanquish UHPLC column and a simple binary gradient， it was demonstrated that ascreening method for 47 compounds within a 4 minute detection window (and a full method cycletime of less than 6 minutes) can be achieved. Using a 500 pL/min flow rate， the system pressure at the start of the gradient was 890 bar， rising toa maximum of 1233 bar during the gradient cycle. The Vanquish UHPLC system is able toroutinely operate at these pressure conditions. Although the data set is complex， the low dispersion of the Vanquish UHPLC system coupled withthe Accucore Vanquish UHPLC column， in combination with the selected-reaction monitoringexperiment， provides sufficient performance to resolve the individual compounds (Figure 1). Time (min) Figure 1： Overlaid selected-reaction monitoring chromatograms showing detection of 47 compounds within a4 minute detection window 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. Figures 2 to 4 show the SRM chromatograms of compounds that demonstrate this capability.Each figure represents data from two isobaric compounds. Figure 2 relates to compounds with molecular weight of 208 u and without common product ions，the chromatograms have a single peak. Figures 3 and 4 relate to compounds with a molecular weight of 300 u and 342 u，respectively.In this situation the compound pairs have common precursor and product ions so multiple peaksare observed but are able to be individually resolved in time. Without this extra chromatographic resolution the identification of these compounds in a screeningset would be compromised as the signals would appear at the same place. Figure 2： Chromatographic resolution of N-desmethyltapentadol and MDEA with common precursor ion(208 u) and different product ions 1.85 Figure 3： Chromatographic resolution of codeine and hydrocodone with common precursor (300 u) andproductions Figure 4： Chromatographic resolution of naltrexone and 6-acetylcodeine with common precursor (342 u) andproduct ions Conclusion This application note demonstrates the advantages of using the Accucore Vanquish C18 1.5 pimUHPLC column and Vanquish UHPLC system. This solution： Delivers a rapid screening method for 47 drugs of abuse Provides method time of less than 6 minutes Exploits the high pressure capabilities of the Vanquish UHPLC system Supports separation of isobaric compounds prior to MS detection [1] McHale， K.， Thermo Scientific， Quantitative LC-MS Screening for Illicit Drugs Using UltrahighResolution Mass Analysis and Accurate Mass Confirmation， Thermo Scientific ApplicationNote (2010) AN63166b_E08/10S. 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