LC/MS/MS Analysis of Gabapentin, Benzodiazepines, and Opiates

Presentation OutlineLC/MS/MS Analysis ofGabapentin,LC/MS/MS Benzodiazepines,Introduction andApplicationsand OpiatesBrian NiesLC/MS Product SpecialistVarian Inc.Winter CAT MeetingRedondo Beach CaliforniaFebruary 6 th , 2008 Introduction to LC/MS Polarity, MW, Volatility Instrumentation Components MS/MS Applications Gabapentin Benzodiazepines Opiates Amphetamines Carboxy-THCPolarity, MW and VolatilityLC/MS Triple Quad ComponentsAutoSamplerData SystemOverlap: GC/MSor LC/MSO.D. Sparkman and F.E. Klink, "Chromatography/Mass Spectrometry: Principlesand Practice", American Chemical Society Continuing Education Courses, 2001.Solvent DeliverySystemMS

IonizationSourceDetectorLC/MS/MS AnalyzerQ1Mass AnalyzerVacuum SystemQ3Mass AnalyzerQ2Collision CeMS/MS what’s that? MS/MS is when you have more than one step ofmass filtration/separation and an additional stepof fragmentation. MS/MS is used to give an additional level ofconfirmation to an analysis or when very specificresults are required. MS/MS can also help determine the molecularstructure of a molecule.Q2- CurvedCollision CellTriple Quad SRMVarian MS Quad: Strong PointsUnique Curved (180°) Collision CellQ1MRM or SRMIonSourceQ1Q3First transitionQ1Q2- CurvedCollision CellExample with a second transition (different precursor and fragment ions)Q3Q2Q1Q2- CurvedCollision CellQ3IonsMRM - often used to describe “Multiple” Reaction Monitoring.SRM – preferred term. Selected Reaction Monitoring (not single).Relating to data acquired from specific product ions correspondingto m/z selected precursor ions.(IUPAC recommendations, www.msterms.com).DetectorQ3

MSMS Using a Curved Collision CellIonSourceQ1• Increased S/N− removal of photons, metastable ions& high energy charged droplets fromion detectorQ2NeutralNoise notdeflectedWhy MS/MSWhy MS/MS for LC/MS?for LC/MS?IonsDetectorQ3Limitation of API Interfaces Atmospheric Pressure Ionization (API) dominate LC/MS Electrospray (ES, ESI, APESI) Chemical Ionization (APCI) Photoionization (APPI) All API mechanisms are “soft” Minimal fragment ions formed during ionizationTypically only molecular ion informationPseudo- or quasi-molecular ion(s) Minimal qualitative information for confirmationAdduct ions (Na, K, etc) are not useful forconfirmation1005077109141195EI Full Scan MS of ReserpineEI-GC/MS produces a wide range of ions“Information Rich”212226251265301359 381040 90 140 190 240 290 340 390 440 490 540 590(mainlib) Reserpine395HNOONO413 448608Electron Ionization spectrum from NIST; probe sample introductionOOOOOO

ESI (+) Full Scan MS of ReserpineESI (+) MS/MS of ReserpineAPI-LC/MS creates very few ionsLimited information(M+H) +After MS/MS dissociationMore ions = more informationNaadductProduct Ion Scan SpectrumAdvantages of MS/MSMS/MS offers increased selectivity for dirtysamples. It helps the users look for smallamounts of sample that would otherwise behidden by all the background noise.ApplicationsSame Molecule, MS Vs. MS/MSMS/MS capabilities cost more due to additional hardware and softwarerequirements in a transmission quadrupole, but often times theperformance is worth the price increase.

Analysis of Gabapentin byLC/MS/MSWhat is Gabapentin?Gabapentin, an analog of the neurotransmitter GABA, is usedas an anticonvulsant in the treatment of epilepsy.It was approved by the FDA as an anti-seizure medication buthas many controversial off-label uses, such as in the treatmentof bipolar disorder, social anxiety disorder, obsessivecompulsivedisorder and insomnia.Gabapentin is also widely used as a pain reliever and was oneof the 50 most prescribed drugs in the United States in 2003due to its mild side-effect profile.Gabapentin may increase the effect of other drugs such asantidepressants, alcohol and pain relievers.Gabapentin StructureGabapentin Extraction MethodologyChemical structure of Gabapentin,(1-(aminomethyl)-cyclohexaneacetic acid)Methodology:The extraction of Gabapentin frombiological specimens is performedusing the principles of proteinprecipitation by acetonitrile (withouta clean up step) and detection byLC/MS/MS.Drugs to be determinedby this method:Gabapentin, Baclofen (IS),Carbinoxamine (Alternate IS)Instrumentation:Liquid Chromatograph withTandem Mass Spectrometer-MassSpectrometer DetectorColumn: Varian Pursuit Diphenyl(DP) Column Dimensions: 3um,50mm x 2.0mm Procedure:Accurately measure 1.0 ml ofstandard, quality control and casesamples.Add 10 ul of internal standard (finalconcentration = 10 ug/ml) to eachtube and vortex.Aliquot 50 ul of each standard,quality control and case sampleinto new tubes.Add 400 ul of acetonitrile to eachtube and vortex.Centrifuge for 10 minutes.Decant supernatant to a cleantube. Evaporate the supernatant todryness. Reconstitute the samples with 200ul of Methanol: Water (60:40).Vortex.Transfer the liquid to labeledAutoSampler vials with inserts.

Conditions MS Conditions:Ionisation Mode: ESI positiveScan Time: 1.0 sDrying Gas: 350 ˚C at 25 psiCollision Cell Pressure: 2.0 mTorrCapillaryVoltage PrecursorProductIonsCollisionEnergyCompounds (V) Ion (m/z) (m/z) (V)Gabapentin 65 172 154 -26.50Gabapentin 65 172 137 -29.00Baclofen(IS) 65 214 151 -36.50Gabapentin MS Parameters HPLC Conditions: Column: Pursuit Diphenyl 50mm x2.0mm 3u Guard Column: Pursuit Diphenyl2.0mm I.D., 3u Solvent A: Water Solvent B: Methanol Flow Rate: 200µL/min Injection Volume: 25µL LC Program:%ATime (min)%B 0:00 60 40 1:001:306054095 3:30 5 95 3:35 60 40 5:00 60 40kCounts 1.0 ug ml std.xms172.0>137.01.0 ug ml std.xms500 172.0>154.040030020010001.0 ug/ml extracted Gabapentin (left); Baclofen – IS(right)0.5 1.0 1.5 2.0 2.5minutesMCounts1.000.750.500.250.00BACLOFEN (IS) 1.0 ug mlstd.xms 151.0(214.0>151.0)0.5 1.0 1.5 2.0 2.5minutesGabapentin Calibration Curve (1.0 ug/ml – 25 ug/ml)Gabapentin ConclusionG A B A P E N T I NC u r v e F i t : L i n e a r , O r i g i n : F o r c e , W e i g h t : N o n e R e s p . F a c t . R S D : 6 . 9y = + 1 . 0 7 0 8 0 6 xC o e f f . D e t . ( r 2 ) : 0 . 9R e p l i c a t e s1 1 1 1 1 12 . 52 . 01 . 51 . 0Peak Size / PS Std.0 . 5LC/MS/MS is utilized in the analysis of gabapentin because itallows for a simple, cost-effective clean up, requires noderivatization of the drug and provides accurate results withexcellent sensitivity.This application demonstrates that the Varian triple quadrupoleLC/MS/MS can identify and quantify Gabapentin in postmortemspecimens with the use of two MRM transitions.Varian Inc. would like to provide a special thank you to SaraKegler and Dan Anderson from Los Angeles County Departmentof Coroner for the validation of both the extraction procedureand the instrument parameters.0 . 00 . 5 1 . 0 1 . 5 2 . 0 2 . 5A m t / M L A m o u n t / . S t d . ( U G )RSD = 6.9%; r2 = 0.999

Introduction to BenzodiazepinesAnalysis of BenzodiazepinesbyLC/MS/MS Benzodiazepines are central nervous systemdepressants. The first benzodiazepine, chlordiazepoxide (Librium®)was discovered in 1954 by the Austrian scientist. In 1963 approval for use was given to diazepam(Valium®) - a simplified version of Librium - primarily tocounteract anxiety symptoms. Currently, fifteen members of this group are marketed inthe United States, and about 20 additionalbenzodiazepines are marketed in other countries.Benzodiazepine Experimental ConditionsCapillary Q1 Q3 Collision AnalyteCID Energy Triple Quadrupole LC/MS/MS ESI Positive LC Conditions: Column: Varian C18 Taxsil 100X2mm,3u Flow Rate: 0.3 mL/min A: 5mM Ammonium Formate B: ACN Time (min) %A %B 0:00 70 30 0:30 70 3010:00 50 5010:30 70 30 15:00 70 30 Injection Volume, 10 uL Mass Spec conditions: ESI Positive Mode Argon pressure: 2.0 mtorr Q1/Q3 peak width: 1.0/1.0amu Samples Protein PrecipitationExtraction Method Complex Matrix Reconstituted in 50:50MeOH:WaterBenzodiazepineMass SpecDetails+ 73.0Volts 271.0 140.0 -24.0Volts Nordiazepam+ 73.0Volts 271.0 165.0 -24.0Volts+ 70.0Volts 285.0 193.0 -24.0Volts Diazepam+ 70.0Volts 285.0 154.0 -19.5Volts+ 70.0Volts 286.0 222.0 -18.5Volts 7-Aminoclonazepam+ 70.0Volts 286.0 121.0 -23.0Volts+ 61.0Volts 287.0 241.0 -10.5Volts Oxazepam+ 61.0Volts 287.0 269.0 -12.0Volts+ 69.0Volts 290.0 226.0 -18.5Volts D-4 7-Aminoclonazepam+ 69.0Volts 290.0 121.0 -23.0Volts+ 69.0Volts 290.1 198.0 -24.0Volts D-5 Diazepam+ 69.0Volts 290.1 154.0 -19.5Volts+ 72.0Volts 292.0 246.0 -10.5Volts D-5 Oxazepam+ 72.0Volts 292.0 274.0 -12.0Volts+ 60.0Volts 301.0 255.0 -18.5Volts Temazepam+ 60.0Volts 301.0 283.0 -10.5Volts+ 80.0Volts 309.0 281.0 -23.5Volts Alprazolam+ 80.0Volts 309.0 205.0 -36.5Volts+ 85.0Volts 314.1 286.0 -18.5Volts D-5 Alprazolam+ 85.0Volts 314.1 268.0 -22.0Volts+ 84.0Volts 316.0 270.0 -20.5Volts Clonazepam+ 84.0Volts 316.0 214.0 -36.0Volts+ 57.0Volts 321.0 275.0 -18.0Volts Lorazepam+ 57.0Volts 321.0 303.0 -16.0Volts

Representative Calibration Curves (10-1000ng/mL)Chromatogramof Benzo Mix,250 ng/mLCalibratorMCounts271.0>140.0 [-24.0V]100MCounts285.0>193.0 [-24.0V]500MCounts286.0>222.0 [-18.5V]500MCounts287.0>241.0 [-10.5V]0.0MCounts290.0>226.0 [-18.5V]20MCounts290.1>198.0 [-24.0V]200MCounts292.0>246.0 [-10.5V]40MCounts301.0>255.0 [-18.5V]200MCounts309.0>281.0 [-23.5V]400MCounts314.1>286.0 [-18.5V]0MCounts316.0>270.0 [-20.5V]40MCounts321.0>275.0 [-18.0V]4NordiazepamDiazepam7-AminoclonazepamOxazepamD-4 7-AminoclonazepamD-5 DiazepamD-5 OxazepamTemazepamAlprazolamD-5 AlprazolamClonazepamLorazepam7-AminoclonazepamWeight: None Curve Fit: Linear, Origin: Include, Resp. Fact. RSD: 8.179%y = +1.058993x -0.006779Coeff. Det.(r2): 0.9993Replicates32 2 2 2 2 25432Peak Size / PS Std.101 2 3 4 5Amount / Amt. Std. (ng/mL)ClonazepamInclude, Weight: None Curve Fit: Linear, Origin: Resp. Fact. RSD: 5.639%y = +1.054153x +0.013111Coeff. Det.(r2): 0.9978Replicates32 2 2 2 2 25432Peak Size / PS Std.1R(2) = 0.999RSD = 8.17R(2) = 0.997RSD = 5.6402.5 5.0 7.5 10.0 12.5 minutesSeg 1, Time: 0.05-15.10, Scan Functions: 282031 4107 6182 8257 10333 Scans01 2 3 4 5Amount / Amt. Std. (ng/mL)Submitted Samples Extracted from Plasma matrixAlternative Benzodiazepine and Method OpiatesUsingUsing the the 320MS Vial 01 Opiate Calibrator (500ng/mL) Codeine Hydrocodone Hydromorphone Morphine Oxycodone Vail 02 Unknown Vial 03 Opiate Internal Stds. (2500ng/mL) Codeine D3 Hydrocodone D6 Hydromorphone D6 Morphine D3 Oxycodone D6 Vial 4 Benzo Internal Stds. (250ng/mL) Nordiazepam D5 Alprazolam D5 Hydroxyalprazolam D5 Vial 5 Benzo Calibrator (250 ng/mL) Nordiazepam D5 Alprazolam D5 Hydroxyalprazolam Nordiazepam Alprazolam Hydroxyalprazolam D5 Flurazepam Oxyazepam Lorazepam Clonazepam Desalkylflurazepam Temazepam Diazepam Vials 6 and 7 Unknowns

320-MS LC SystemsBreakdown CurvesHydroxyalprazolamHydrocodone320-MS212-LCProStar 430320-MS212-LCHTS PALBenzodiazepines MethodBenzo Three Ion Monitoring Mobile Phase: 0.1% Formicaq/MeOH Nebulizer Gas: N 2 @ 30psi Drying gas: N 2 @ 24psi (350C) Flow rate: 200 uL/min Column: Pursuit XRS Diphenyl 100x 4.6mm 3umExample:Hydroxyalprazolam325>297 (Quant. Ion)325>216 (Qualifier)325>279 (Qualifier)

Extracted Spiked Plasma Benzo MRMChromatograms (10 ng/mL)Benzo resultsOpiates MethodExtracted Spiked Plasma Opiate MRMChromatograms (50ng/mL)Mobile Phase: 0.1% Formicaq/MeOHNebulizer Gas: N 2 @ 30psiDrying gas: N 2 @ 24psi (350C)Flow rate: 200 uL/minColumn: MetaChem Taxil 100 x2mm 3um

Opiate resultsCustom ReportsBenzodiazepine and OpiatesSummaryExcellent sensitivityQuantitation limits will be below 10ng/mL (50ng for Opiates)Excellent quantitation can be achieved using a select fewinternal standardsBoth LC programs have a 15 minute run time.The additional sensitivity of 320ms allows for three ionmonitoring of each analyte.Analysis of Amphetamines byLC/MS/MS

Replicates 93 3 3 3 33002502001501005002 50 0 5 0 00 75 00Amount / Amt. Std.Rep lic ate s 24M15 012 510 07550250.999900 0.999925 0.999950 0.999975 1.000000 1.000025 1.000050 1.000075AmountAmphetamines Experimental ConditionsMethamphetamine: MS/MS BreakdownMethamphetamine Calibration CurveChromatographic data of AmphetaminesCalibration Curve ReportFile: e:\norchem\amps\amphetaminesdata multiplier2.mthDetector: 1200 Mass Spec, Address: 42Calibration Curve ReportFile: e:\norchem\amps\amphetaminesdata multiplier2.mthDetector: 1200 Mass Spec, Address: 42MethamphetamineC urve Fit: Linear, Origin: Ignore, Weight: None Resp. Fact. R SD : 5.318%y = +0.031914x +0.257271Coeff. Det.(r2): 0.997419Methamphetamine d5C urve Fit: Linear, Origin: Include (Ignore), W eight: N one Resp. Fact. R SD : 0.0000%y = +1.000000x +0.0C o eff. D et.( r2) : 1.00000 0Peak Size / PS Std.Peak SizeMethamphetamine 5-10,000 ng/mLMethamphetamine d5 ISSpiked: 50 ng/mL in urine

Amphetamine Summary Excellent sensitivity at the level of 5ng/mL Good separation from a column packed with afluorinated phenyl phase Detection limits will be at least the low ppb levels for allcompounds All 6 drugs can be run simultaneously in a 6 min. run Good linearity of calibration curves ranging from 5-10,000 ng/mLAnalysis of Carboxy-THCbyLC/MS/MSCarboxy -THC Experimental ConditionsChromatograms – Carboxy - THC ESI Negative. LC Conditions: Column: Varian Pursuit Diphenyl50X2mm, 3μ Flow Rate: 0.2 mL/min A: Water B: Methanol Time %B 0:00 40 0:30 40 1:00 95 3:00 95 3:01 40 6:30 40 Injection Volume, 20 μL Mass Spec conditions: Argon pressure: 2.0 mtorr Q1/Q3 peak width: 1.0/1.0amu Transitions: 343.50 -> 299 CE 17.5 352.50 -> 308 CE 20.5 SamplesIn a complicated matrixsolutionStandard Solutions preparedby serial dilutions in DI water50 μL IS added to 1 mL ofsampleCarboxy-THC – 10 ng/mLCarboxy-THC – 1 ng/mL

Carboxy -THC Calibration Curve ReportCarboxy -THC Sample Report ExampleCarboxy-THC 1-1000ng/mLCarboxy-THC d9 - ISCarboxy -THC Summary Excellent sensitivity at the 1 ng/mL Detection limits in the sub ppb levels Linear calibration curve from 1-1000 ng/mL R 2 value of 0.998 Run time of only 6.5 minutes