High-Throughput Quantitative LC-MS/MS Analysis Of 6 .

Bill Yu, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, San Jose, CAKey WordsOpiates, benzodiazepines, Prelude SPLCGoalDevelop a high-throughput, low solvent consumption, easy-to-runmethod for quantitative forensic analysis of six opiates and fourteenbenzodiazepines in urine.IntroductionAnalyses of opiate and benzodiazepine panels are some ofthe highest-volume applications in forensic toxicologylabs. In order to meet the need for high throughput, a fast,simple, and cost-effective method was developed,consisting of hydrolysis, simple urine dilution, separationby liquid chromatography (LC), and analysis by massspectrometry (MS). The method incorporated theThermo Scientific Prelude SPLC system (Figure 1),which features two independent channels of samplepreparation and liquid chromatography (SPLC). Withthe Prelude SPLC system, LC methods can be executedin parallel with a different method on each channel(Figure 2) or the same method on both channels(Figure 3) and multiplexed into a mass spectrometerFigure 1. Prelude SPLC systemfor serial detection. Serial MS detection of multiplexedmethods improves mass spectrometer utilization time,increases throughput of forensic toxicology laboratories,and reduces analysis cost. The syringe pumps andhigh-pressure, low-volume gradient mixing used inPrelude SPLC system provide enhanced HPLCperformance: improved peak shape and resolution as wellas stable retention times.Conventional LC-MS/MS14 Benzodiazepines(25 ng/mL each)14 BenzodiazepinesInjection 10.01.0Injection 22.03.04.05.06.07.0Injection 38.09.010.011.012.013.0Prelude SPLC multiplexingChannel 114 Benzodiazepines(25 ng/mL each)14 BenzodiazepinesInjection 10.01.0Injection 2Injectio2.03.04.05.06.07.0Injection 38.01.010.011.02.03.04.012.0113.06 OpiatesInjection 10.09.06 Opiates(10 ng/mL each)Channel 2Injection 25.06.07.08.09.010.011.012.013.0Acquisition of relevent MS dataFigure 2. Parallel analysis of 6 opiates (10 ng/mL) and 14 benzodiazepines (25 ng/mL) in multiplexed modeAppli cat i on N ote 5 8 8High-Throughput QuantitativeLC-MS/MS Analysis of 6 Opiatesand 14 Benzodiazepines in Urine

2Prelude SPLC Multiplexing - Same Method on Both ChannelsChannel 16 Opiates(10 ng/mL each)Injection 11.00.06 Opiates6 Opiates6 OpiatesInjection 23.02.04.05.06.0Injection 37.08.09.010.011.012.013.010.011.012.013.0Channel 2Injection 11.00.03.02.0Injection 24.05.06.07.08.09.0Acquisition of relevent MS dataFigure 3. Analysis of six opiates (10 ng/mL) using both channels in multiplexed modeExperimentalTable 2. SRM transitions for benzodiazepines methodSample PreparationTables 1 and 2 contain the lists of opiates andbenzodiazepines analyzed. Sample preparation consistedof glucuronide hydrolysis followed by dilution. For eachsample, a 200 µL aliquot of urine was spiked with 10 µLof internal standards solution and 100 µL ofb-glucuronidaze enzyme in an ammonium acetate buffer(pH 5.0). The samples were incubated at 60 C for2 hours. A 200 µL aliquot of methanol was added to eachsample to stop enzymatic reaction. Samples were cooled,centrifuged, and diluted 20 times with deionized water.Then, 20 µL of sample was injected into the liquidchromatograph-mass spectrometer (LC-MS) system.Table 1. SRM transitions for opiates methodCompoundCompoundPrecursorIon m/zQuantifierIon m/zQualifierIon sorIon m/zQuantifierIon m/zQualifier 171.1Midazolam-d4330.1295.1253.1Morphine

Liquid ChromatographyChromatographic separations were performed with aPrelude SPLC system by direct injections ontoThermo Scientific Accucore PFP 50 x 2.1 mm, 2.6 µmanalytical columns. The columns were maintained atroom temperature. Mobile phases A and B consisted of10 mM ammonium formate with 0.1% formic acid inwater and methanol, respectively. Mobile phase usage wasabout 3.8 mL per sample. The total gradient run time was5.3 min for opiates analysis (Figure 4) and 6 min forbenzodiazepines analysis (Figure 5). The data acquisitionwindows were 2 min and 2.8 min for opiates andbenzodiazepines, respectively.Figure 4. LC gradient for opiates analysisFigure 5. LC gradient for benzodiazepines analysisMass SpectrometryMS analysis was carried out on a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometerequipped with a heated electrospray ionization (HESI-II)probe. The mass spectrometer was operated in selectedreaction monitoring (SRM) mode. Two SRM transitionswere collected for each analyte and each internal standard(Tables 1 and 2) to calculate the ion ratio.ValidationStandard curves were prepared by fortifying pooled blankhuman urine with analytes. Quality control (QC) sampleswere prepared in a similar manner at concentrationscorresponding to the low (LQC), middle (MQC), andhigh (HQC) ranges of the calibration curve. Intra-runprecisions were determined by processing six replicates ofeach QC level along with a calibration curve on threedifferent days. Matrix effects were investigated byanalyzing seven donated urine samples spiked atconcentrations of 27.5 ng/mL for opiates and 50 ng/mLfor benzodiazepines. The method performance wascompared with method validated in a forensic toxicologylab by analyzing the same donor samples. Methodvalidation experiments were run by executing opiates andbenzodiazepines methods in parallel on two channels inmultiplexed mode.3

4Results and DiscussionOpiates AnalysisThe limits of quantitation were 10 ng/mL and calibrationranges were 10–6000 ng/mL for all opiates. Figure 6shows representative calibration curves for selectedopiates. Figure 7 shows representative chromatograms at10 ng/mL for all opiates tested. Intra- and inter-assayquality control statistics shown in Table 3 demonstrate themethod to be reproducible across the calibration range forthe opiates. Limited matrix effects were seen, and thosewere largely mediated by deuterated internal standards(Table 4). The data collected with this method correlatedwell with data collected using an LC/MS methodpreviously validated in a collaborating laboratory (Figure 8).Figure 6. Calibration curves for selected opiatesFigure 7. Chromatograms of the lowest opiates calibrationstandard (10 ng/mL)

5400002R 0.99743500025000Validated Method (ng/mL)Validated Method rphone500050000100020003000Prelude SPLC-Based Method elude SPLC-Based Method (ng/mL)Figure 8. Data correlation between Prelude SPLC-based opiates method and a previously validated LC/MS methodTable 3. Intra- and inter-assay precision for opiates analysesPrecision % LQCMQCHQC 7.0 2.8 2.38.32.53.3Hydromorphone 3.7 1.8 1.74.72.22.7Oxymorphone 5.9 4.8 4.99.84.99.8Codeine 8.2 11 2.28.24.83.0Hydrocodone 4.7 3.8 2.84.73.94.2Oxycodone 7.4 3.9 2.87.13.83.6Table 4. Results of matrix effect experiment showing percent recovery of opiates in spiked urineUrine Lot#% 03792.097.8108109100103

6Benzodiazepines AnalysisThe limits of quantitation were 25 ng/mL and calibrationranges were 25–2000 ng/mL for all benzodiazepines.Figure 9 shows representative calibration curves forselected benzodiazepines. Figure 10 shows representativechromatograms at 25 ng/mL for all benzodiazepines tested.Intra- and inter-assay quality control statistics shown inTable 5 demonstrate the method to be reproducible acrossthe calibration range for these benzodiazepines. Use ofdeuterated internal standard eliminated the small matrixeffects we experienced with the method (Table 6). The datacollected with this method correlated well with datacollected using an LC/MS method previously validated in acollaborating laboratory (Figure 11).Figure 9. Calibration curves for selected 01.52.02.50.51.0Time (min)Figure 10. Chromatogram of the lowest benzodiazepines calibration standard (10 ng/mL)1.5Time (min)2.02.5

7400R 2 0.9377350Validated Method zepamTemazepam500050100150200250300350400Prelude SPLC-Based Method (ng/mL)Figure 11. Data correlation between Prelude SPLC-based benzo-diazepines method and a previously validatedLC/MS methodTable 5. Intra and inter-assay precision for benzodiazepines analysesCompoundPrecision % lflurazepam 10 3.38.94.27-Aminoclonazepam 2.5 2.72.52.07-Aminoflunitrazepam 3.6 3.23.12.97-Aminonitrazepam 2.7 3.62.42.7a-Hydroxyalprazolam 5.8 4.15.84.5a-Hydroxytriazolam 5.9 4.17.23.8Alprazolam 5.2 2.13.52.3Desalkyflurazepam 5.3 5.93.62.3Diazepam 2.8 3.03.12.2Lorazepam 5.3 4.56.73.3Midazolam 1.2 5.42.81.6Nordiazepam 4.0 4.55.12.5Oxazepam 3.3 3.23.33.9Temezepam 5.3 3.14.33.6

Table 6. Results of matrix effect experiment showing percent recovery of benzodiazepines in spiked urine% 8.510111293.0107UrineLot#% 11195.7103ConclusionAcknowledgementUsing the Prelude SPLC system, high-throughput, costefficient solutions were developed for forensic analysis ofopiates and benzodiazepines in urine. The methods metindustry requirements for precision, accuracy, androbustness. Implementation of the method on a PreludeSPLC simplified the work flow and resulted in a 40–60%reduction of solvent usage due to the ability of the system toutilize high efficiency, small diameter columns. The mobilephase volumes in developed methods were approximately3.8 mL per sample, which reduced cost of reagents andwaste disposal. Multiplexing into a single mass spectrometerincreased MS utilization and reduced overall systemhardware costs relative to two independent LC-MS systems.The Prelude SPLC system makes multiplexing of twodifferent methods, with or without on-line sample prep,possible and enabled a throughput of 480 samples in24 hours. The implementation of methods was facilitated bythe many ease-of-use features incorporated into the system.Thanks to Kent Johnson, Director of Toxicology atPacific Hospital of Long Beach, CA for providing donorurine samples and data collected with their validatedLC-MS method.For Forensic Toxicology use only.www.thermoscientific.com 2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a registered trademark of the International Organization for Standardization(Organisation Internationale De Normalization). All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Thisinformation is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of theseproducts in any manners that might infringe 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.Africa-Other 27 11 570 1840Australia 61 3 9757 4300Austria 43 810 282 206Belgium 32 53 73 42 41Canada 1 800 530 8447China 86 10 8419 3588Denmark 45 70 23 62 60Europe-Other 43 1 333 50 34 0Finland 358 9 3291 0200France 33 1 60 92 48 00Germany 49 6103 408 1014India 91 22 6742 9494Italy 39 02 950 591Japan 81 45 453 9100Latin America 1 561 688 8700Middle East 43 1 333 50 34 0Netherlands 31 76 579 55 55New Zealand 64 9 980 6700Norway 46 8 556 468 00Russia/CIS 43 1 333 50 34 0South Africa 27 11 570 1840Thermo Fisher Scientific,San Jose, CA USA isISO 9001:2008 Certified.Spain 34 914 845 965Sweden 46 8 556 468 00Switzerland 41 61 716 77 00UK 44 1442 233555USA 1 800 532 4752AN63754 E 10/13SAppli cat i on N ote 5 8 8UrineLot#

High-Throughput Quantitative LC-MS/MS Analysis of 6 Opiates and 14 Benzodiazepines in Urine Bill Yu, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, San Jose, CA Application Note 588 Key Words Opiates, benzodiazepines, Prelude SPLC Goal Develop a high-throughput, low solvent consumption, easy-to-run