LC/MS/MS Analysis of Gabapentin, and Opiates. Applications. Presentation Outline. Polarity, MW and Volatility LC/MS Triple Quad Components

Transcription

1 Presentation Outline LC/MS/MS Analysis of Gabapentin, LC/MS/MS Benzodiazepines, Introduction and Applications and Opiates Brian Nies LC/MS Product Specialist Varian Inc. Winter CAT Meeting Redondo Beach California February 6 th, 28 Introduction to LC/MS Polarity, MW, Volatility Instrumentation Components MS/MS Applications Gabapentin Benzodiazepines Opiates Amphetamines Carboxy-THC Polarity, MW and Volatility LC/MS Triple Quad Components AutoSampler Data System Overlap: GC/MS or LC/MS O.D. Sparkman and F.E. Klink, "Chromatography/Mass Spectrometry: Principles and Practice", American Chemical Society Continuing Education Courses, 21. Solvent Delivery System MS

2 Ionization Source Detector LC/MS/MS Analyzer Q1 Mass Analyzer Vacuum System Q3 Mass Analyzer Q2 Collision Ce MS/MS what s that? MS/MS is when you have more than one step of mass filtration/separation and an additional step of fragmentation. MS/MS is used to give an additional level of confirmation to an analysis or when very specific results are required. MS/MS can also help determine the molecular structure of a molecule. Q2- Curved Collision Cell Triple Quad SRM Varian MS Quad: Strong Points Unique Curved (18 ) Collision Cell Q1 MRM or SRM Ion Source Q1 Q3 First transition Q1 Q2- Curved Collision Cell Example with a second transition (different precursor and fragment ions) Q3 Q2 Q1 Q2- Curved Collision Cell Q3 Ions MRM - often used to describe Multiple Reaction Monitoring. SRM preferred term. Selected Reaction Monitoring (not single). Relating to data acquired from specific product ions corresponding to m/z selected precursor ions. (IUPAC recommendations, Detector Q3

3 MSMS Using a Curved Collision Cell Ion Source Q1 Increased S/N removal of photons, metastable ions & high energy charged droplets from ion detector Q2 Neutral Noise not deflected Why MS/MS Why MS/MS for LC/MS? for LC/MS? Ions Detector Q3 Limitation 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 ionization Typically only molecular ion information Pseudo- or quasi-molecular ion(s) Minimal qualitative information for confirmation Adduct ions (Na, K, etc) are not useful for confirmation EI Full Scan MS of Reserpine EI-GC/MS produces a wide range of ions Information Rich 212 O O O 5 O O O O O (mainlib) Reserpine Electron Ionization spectrum from NIST; probe sample introduction O HN N

4 ESI (+) Full Scan MS of Reserpine ESI (+) MS/MS of Reserpine API-LC/MS creates very few ions Limited information (M+H) + After MS/MS dissociation More ions = more information Na adduct Product Ion Scan Spectrum Advantages of MS/MS MS/MS offers increased selectivity for dirty samples. It helps the users look for small amounts of sample that would otherwise be hidden by all the background noise. Applications Same Molecule, MS Vs. MS/MS MS/MS capabilities cost more due to additional hardware and software requirements in a transmission quadrupole, but often times the performance is worth the price increase.

5 Analysis of Gabapentin by LC/MS/MS What is Gabapentin? Gabapentin, an analog of the neurotransmitter GABA, is used as an anticonvulsant in the treatment of epilepsy. It was approved by the FDA as an anti-seizure medication but has many controversial off-label uses, such as in the treatment of bipolar disorder, social anxiety disorder, obsessivecompulsive disorder and insomnia. Gabapentin is also widely used as a pain reliever and was one of the 5 most prescribed drugs in the United States in 23 due to its mild side-effect profile. Gabapentin may increase the effect of other drugs such as antidepressants, alcohol and pain relievers. Gabapentin Structure Gabapentin Extraction Methodology Chemical structure of Gabapentin, (1-(aminomethyl)-cyclohexaneacetic acid) Methodology: The extraction of Gabapentin from biological specimens is performed using the principles of protein precipitation by acetonitrile (without a clean up step) and detection by LC/MS/MS. Drugs to be determined by this method: Gabapentin, Baclofen (IS), Carbinoxamine (Alternate IS) Instrumentation: Liquid Chromatograph with Tandem Mass Spectrometer-Mass Spectrometer Detector Column: Varian Pursuit Diphenyl (DP) Column Dimensions: 3um, 5mm x 2.mm Procedure: Accurately measure 1. ml of standard, quality control and case samples. Add 1 ul of internal standard (final concentration = 1 ug/ml) to each tube and vortex. Aliquot 5 ul of each standard, quality control and case sample into new tubes. Add 4 ul of acetonitrile to each tube and vortex. Centrifuge for 1 minutes. Decant supernatant to a clean tube. Evaporate the supernatant to dryness. Reconstitute the samples with 2 ul of Methanol: Water (6:4). Vortex. Transfer the liquid to labeled AutoSampler vials with inserts.

6 Conditions MS Conditions: Ionisation Mode: ESI positive Scan Time: 1. s Drying Gas: 35 C at 25 psi Collision Cell Pressure: 2. mtorr Capillary Voltage Precursor Product Ions Collision Energy Compounds (V) Ion (m/z) (m/z) (V) Gabapentin Gabapentin Baclofen (IS) Gabapentin MS Parameters HPLC Conditions: Column: Pursuit Diphenyl 5mm x 2.mm 3u Guard Column: Pursuit Diphenyl 2.mm I.D., 3u Solvent A: Water Solvent B: Methanol Flow Rate: 2µL/min Injection Volume: 25µL LC Program: %A Time (min) %B : 6 4 1: 1: : : : 6 4 kcounts 1. ug ml std.xms 172.> ug ml std.xms > ug/ml extracted Gabapentin (left); Baclofen IS (right) minutes MCounts BACLOFEN (IS) 1. ug ml std.xms 151. (214.>151.) minutes Gabapentin Calibration Curve (1. ug/ml 25 ug/ml) Gabapentin Conclusion G A B A P E N T I N C 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. 9 y = x C o e f f. D e t. ( r 2 ) :. 9 R e p l i c a t e s Peak Size / PS Std.. 5 LC/MS/MS is utilized in the analysis of gabapentin because it allows for a simple, cost-effective clean up, requires no derivatization of the drug and provides accurate results with excellent sensitivity. This application demonstrates that the Varian triple quadrupole LC/MS/MS can identify and quantify Gabapentin in postmortem specimens with the use of two MRM transitions. Varian Inc. would like to provide a special thank you to Sara Kegler and Dan Anderson from Los Angeles County Department of Coroner for the validation of both the extraction procedure and the instrument parameters A m t / M L A m o u n t /. S t d. ( U G ) RSD = 6.9%; r2 =.999

7 Introduction to Benzodiazepines Analysis of Benzodiazepines by LC/MS/MS Benzodiazepines are central nervous system depressants. 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 to counteract anxiety symptoms. Currently, fifteen members of this group are marketed in the United States, and about 2 additional benzodiazepines are marketed in other countries. Benzodiazepine Experimental Conditions Capillary Q1 Q3 Collision Analyte CID Energy Triple Quadrupole LC/MS/MS ESI Positive LC Conditions: Column: Varian C18 Taxsil 1X2mm, 3u Flow Rate:.3 ml/min A: 5mM Ammonium Formate B: ACN Time (min) %A %B : 7 3 : : 5 5 1: : 7 3 Injection Volume, 1 ul Mass Spec conditions: ESI Positive Mode Argon pressure: 2. mtorr Q1/Q3 peak width: 1./1. amu Samples Protein Precipitation Extraction Method Complex Matrix Reconstituted in 5:5 MeOH:Water Benzodiazepine Mass Spec Details + 73.Volts Volts Nordiazepam + 73.Volts Volts + 7.Volts Volts Diazepam + 7.Volts Volts + 7.Volts Volts 7-Aminoclonazepam + 7.Volts Volts + 61.Volts Volts Oxazepam + 61.Volts Volts + 69.Volts Volts D-4 7-Aminoclonazepam + 69.Volts Volts + 69.Volts Volts D-5 Diazepam + 69.Volts Volts + 72.Volts Volts D-5 Oxazepam + 72.Volts Volts + 6.Volts Volts Temazepam + 6.Volts Volts + 8.Volts Volts Alprazolam + 8.Volts Volts + 85.Volts Volts D-5 Alprazolam + 85.Volts Volts + 84.Volts Volts Clonazepam + 84.Volts Volts + 57.Volts Volts Lorazepam + 57.Volts Volts

8 Representative Calibration Curves (1-1ng/mL) Chromatogram of Benzo Mix, 25 ng/ml Calibrator MCounts271.>14. [-24.V] 1 MCounts285.>193. [-24.V] 5 MCounts286.>222. [-18.5V] 5 MCounts287.>241. [-1.5V]. MCounts29.>226. [-18.5V] 2 MCounts29.1>198. [-24.V] 2 MCounts292.>246. [-1.5V] 4 MCounts31.>255. [-18.5V] 2 MCounts39.>281. [-23.5V] 4 MCounts314.1>286. [-18.5V] MCounts316.>27. [-2.5V] 4 MCounts321.>275. [-18.V] 4 Nordiazepam Diazepam 7-Aminoclonazepam Oxazepam D-4 7-Aminoclonazepam D-5 Diazepam D-5 Oxazepam Temazepam Alprazolam D-5 Alprazolam Clonazepam Lorazepam 7-Aminoclonazepam Weight: None Curve Fit: Linear, Origin: Include, Resp. Fact. RSD: 8.179% y = x Coeff. Det.(r2):.9993 Replicates Peak Size / PS Std Amount / Amt. Std. (ng/ml) Clonazepam Include, Weight: None Curve Fit: Linear, Origin: Resp. Fact. RSD: 5.639% y = x Coeff. Det.(r2):.9978 Replicates Peak Size / PS Std. 1 R(2) =.999 RSD = 8.17 R(2) =.997 RSD = minutes Seg 1, Time: , Scan Functions: Scans Amount / Amt. Std. (ng/ml) Submitted Samples Extracted from Plasma matrix Alternative Benzodiazepine and Method OpiatesUsing Using the the 32MS Vial 1 Opiate Calibrator (5 ng/ml) Codeine Hydrocodone Hydromorphone Morphine Oxycodone Vail 2 Unknown Vial 3 Opiate Internal Stds. (25 ng/ml) Codeine D3 Hydrocodone D6 Hydromorphone D6 Morphine D3 Oxycodone D6 Vial 4 Benzo Internal Stds. (25 ng/ml) Nordiazepam D5 Alprazolam D5 Hydroxyalprazolam D5 Vial 5 Benzo Calibrator (25 ng/ml) Nordiazepam D5 Alprazolam D5 Hydroxyalprazolam Nordiazepam Alprazolam Hydroxyalprazolam D5 Flurazepam Oxyazepam Lorazepam Clonazepam Desalkylflurazepam Temazepam Diazepam Vials 6 and 7 Unknowns

9 32-MS LC Systems Breakdown Curves Hydroxyalprazolam Hydrocodone 32-MS 212-LC ProStar MS 212-LC HTS PAL Benzodiazepines Method Benzo Three Ion Monitoring Mobile Phase:.1% Formic aq/meoh Nebulizer Gas: N 3psi Drying gas: N 24psi (35C) Flow rate: 2 ul/min Column: Pursuit XRS Diphenyl 1 x 4.6mm 3um Example: Hydroxyalprazolam 325>297 (Quant. Ion) 325>216 (Qualifier) 325>279 (Qualifier)

10 Extracted Spiked Plasma Benzo MRM Chromatograms (1 ng/ml) Benzo results Opiates Method Extracted Spiked Plasma Opiate MRM Chromatograms (5ng/mL) Mobile Phase:.1% Formic aq/meoh Nebulizer Gas: N 3psi Drying gas: N 24psi (35C) Flow rate: 2 ul/min Column: MetaChem Taxil 1 x 2mm 3um

11 Opiate results Custom Reports Benzodiazepine and Opiates Summary Excellent sensitivity Quantitation limits will be below 1ng/mL (5ng for Opiates) Excellent quantitation can be achieved using a select few internal standards Both LC programs have a 15 minute run time. The additional sensitivity of 32ms allows for three ion monitoring of each analyte. Analysis of Amphetamines by LC/MS/MS

12 Replicates Amount / Amt. Std. Rep lic ate s 24 M Amount Amphetamines Experimental Conditions Methamphetamine: MS/MS Breakdown Methamphetamine Calibration Curve Chromatographic data of Amphetamines Calibration Curve Report File: e:\norchem\amps\amphetaminesdata multiplier2.mth Detector: 12 Mass Spec, Address: 42 Calibration Curve Report File: e:\norchem\amps\amphetaminesdata multiplier2.mth Detector: 12 Mass Spec, Address: 42 Methamphetamine C urve Fit: Linear, Origin: Ignore, Weight: None Resp. Fact. R SD : 5.318% y = x Coeff. Det.(r2): Methamphetamine d5 C urve Fit: Linear, Origin: Include (Ignore), W eight: N one Resp. Fact. R SD :.% y = +1.x +. C o eff. D et.( r2) : 1. Peak Size / PS Std. Peak Size Methamphetamine 5-1, ng/ml Methamphetamine d5 IS Spiked: 5 ng/ml in urine

13 Amphetamine Summary Excellent sensitivity at the level of 5ng/mL Good separation from a column packed with a fluorinated phenyl phase Detection limits will be at least the low ppb levels for all compounds All 6 drugs can be run simultaneously in a 6 min. run Good linearity of calibration curves ranging from 5-1, ng/ml Analysis of Carboxy-THC by LC/MS/MS Carboxy -THC Experimental Conditions Chromatograms Carboxy - THC ESI Negative. LC Conditions: Column: Varian Pursuit Diphenyl 5X2mm, 3μ Flow Rate:.2 ml/min A: Water B: Methanol Time %B : 4 :3 4 1: 95 3: 95 3:1 4 6:3 4 Injection Volume, 2 μl Mass Spec conditions: Argon pressure: 2. mtorr Q1/Q3 peak width: 1./1. amu Transitions: > 299 CE > 38 CE 2.5 Samples In a complicated matrix solution Standard Solutions prepared by serial dilutions in DI water 5 μl IS added to 1 ml of sample Carboxy-THC 1 ng/ml Carboxy-THC 1 ng/ml

14 Carboxy -THC Calibration Curve Report Carboxy -THC Sample Report Example Carboxy-THC 1-1 ng/ml Carboxy-THC d9 - IS Carboxy -THC Summary Excellent sensitivity at the 1 ng/ml Detection limits in the sub ppb levels Linear calibration curve from 1-1 ng/ml R 2 value of.998 Run time of only 6.5 minutes