The Analysis of Drugs of Abuse Using LC/MS

Transcription

1 John Hughes Application Engineer September 16, 23 The Analysis of Drugs of Abuse Using LC/MS Part 1 of a 2-part E-seminar series Life Sciences and Chemical Analysis

2 Presenter John Hughes, Ph.D. John is a mass spectrometry applications consultant with Agilent Technologies, located in Pleasanton, CA. In that role, he is one of 2 applications specialists in North America responsible for applications support for GC/MS and LC/MS systems. He has been with HP and Agilent for 18 years as an MS applications chemist. His particular areas of expertise include applications of mass spectrometry to pharmaceutical chemistry, natural products, synthetic organic chemistry, forensic science, and toxicology. Slide 2

3 Today s Topics: Introduction to Modern LC/MS Cocaine and metabolites in urine Opiates and metabolites in blood Rapid multi-class LC/MS drug screen and confirmation Slide 3 Part 1 of this e-seminar series contains the following topics.

4 Other Topics of Forensic Interest: E-seminars ( click Events) Drugs by LC/MS Part 2: Sept 23: Intro to LC/MS (repeat), LSD, THC, GHB New 5973 Inert: Sept 3 Rapid Drug Screening using RTL GC/MS: Nov13 Products Zorbax HPLC columns: New <2µ high-speed LCMSD-TOF: accurate mass for confirmation Slide 4 Other topics of possible interest to drug laboratories

5 Introduction to API-LC/MS

6 Myths of LC/MS LC/MS is not routine API only produces molecular weight information LC/MS is not sensitive LC/MS is not quantitative LC/MS is not cost-effective Slide 6 The early days of LC/MS used complicated interfaces and difficult techniques like direct-liquid introduction (DLI) and thermospray, which led to a number of beliefs about LC/MS which linger on in lab folklore. The first commercial LC/MS interfaces were on large MS systems of the 7 s and 8 s which could be the size of a large conference table and require a highlytrained, dedicated operator. All of these issues are no longer pertinent to modern LC/MS systems.

7 Modern Benchtop LC/MS Systems Slide 7 Modern benchtop LC/MS systems are no more difficult to use than the benchtop GC/MS systems which have been in routine use in toxicology and crime labs for almost two decades. API LC/MS in some ways is easier than GC/MS. Most of the contaminants which would impair the operation of the MS detector, are vented or left outside the vacuum region of the MS, where they can be easily removed in a few minutes (at least in Agilent systems) by daily and weekly maintenance, without venting the MS. Any competent LC chromatographer can quickly learn to use the LC/MSD as just another LC detector. A competent mass spectrometrist can quickly learn to use the LC/MSD as an MS with many modes of soft ionization, along with a separation mode that works well with a huge variety of analyte classes, polarities and molecular weights.

8 Benefits of LC/MS for the Forensic Scientist Can analyze compounds not amenable to GC without derivatization (polar, thermally labile, non-volatile, high MW) Complements existing LC detectors All the benefits of MS: Independent of a particular functional group Can be used as a compound-specific detector Provides both qualitative and quantitative information Easy-to-use as an integrated system Slide 8 LC/MS has been aggressively pursued by both mass spectrometrists and liquid chromatographers as a "hyphenated" technique for many years. The slide illustrates some of the reasons for the high level of interest in LC/MS.

9 Interfacing HPLC to MS HPLC High pressure liquid phase separation produces high gas load no mass range limitation can use inorganic buffers MS high vacuum required tolerates limited gas load elevated temperatures depends on m/z and analyzer prefers volatile buffers HPLC Interface (Key to Success!) MS Slide 9 LC/MS was developed commercially some years after GC/MS, largely because of the greater difficulty in connecting a separation device with a condensed mobile phase, to a detector which requires gas phase analysis. It was also necessary to develop an ionization technique capable of producing ions from the wide variety of compounds analyzed by HPLC. Lastly, the LC/MS interface needed to be able to handle a wide range of LC flows, mobile phases, and mobile phase modifiers. Atmospheric Pressure Ionization (API) finally has achieved these objectives.

10 Comparison of Ionization Modes

11 API Electrospray LC/MSD HPLC inlet Nebulizer gas inlet Nebulizer Capillary Skimmers Octopole Lenses Quadrupole HED detector Neutral Molecules Analyte Ions Clusters Salts heated N 2 Waste Fragmentation zone (CID) Slide 11 Now lets take a close look at API-electrospray ionization. The API-electrospray process comprises 3 basic steps: (1) nebulization and charging; (2) desolvation; (3) ion evaporation. Nebulization and charging occur as the HPLC effluent with analyte ions in solution emerges from the tip of the nebulizing needle, which is at ground potential, into a semi-cylindrical electrode to which high voltage is applied. The potential difference between the nebulizer and the counter-electrode produces a strong electric field that charges the surface of the emerging liquid and forms a fine spray of charged droplets. Concentric high-pressure gas flow assists the nebulization process. Desolvation: the charged droplets are attracted toward the capillary sampling orifice through a counter flow of heated nitrogen drying gas, which shrinks the droplets and carries away uncharged material. Ion evaporation: The droplets continue to shrink until the repulsive electrostatic (Coulombic) forces exceed the droplet cohesive forces, leading to droplet explosions. This process is repeated until analyte ions are ultimately desorbed into the gas phase, driven by strong electric fields on the surface of the microdroplets. The emerging gas phase ions are then passed through the capillary sampling orifice into the low pressure region of the ion source, and on to the mass analyzer. Electrospray may be though of as ionization followed by ion evaporation.

12 Theory: API Electrospray Charged Droplets Evaporation Analyte Ions Solvent Ion Clusters Salts/Ion pairs Neutrals Rayleigh Limit Reached Coulombic Explosions -- Solvent Ion Cluster Analyte Ion Slide 12 This slide shows the electrospray process at the droplet and ion level. Ions are ejected from the charged droplet as described in the previous slide and notes.

13 APCI LC/MSD Nebulizer Heater Skimmers Lenses HED detector Capillary Octopole Quadrupole [SolvH] A Solv [AH] Corona needle heated N 2 Waste Fragmentation zone (CID) Slide 13 The HP LC/MSD APCI interface is used for low to medium polarity analytes. The APCI process generates ions by first nebulizing the liquid eluate into small droplets, followed by evaporating the droplets to produce gas phase analyte and mobile phase molecules. The gas phase analyte is then ionized by gas phase CI via proton addition, proton abstraction, or by electron capture processes. The corona discharge needle serves as a charge source. Analyte can be ionized directly or by interaction with ions formed from the LC mobile phase. Just as in the API-electrospray design, the APCI inlet is positioned orthogonally to the inlet of the capillary, uses the same nebulizer design, and takes advantage of the drying gas heater design. All of this results in low noise, high signal, and maximum system uptime at high HPLC flow rates. The externally removable corona discharge needle can be easily replaced without venting the vacuum system or opening the spray chamber. APCI can be thought of as evaporation followed by ionization.

14 Theory: Atmospheric Pressure Chemical Ionization (APCI) Aerosol droplets containing analyte Vapor Corona discharge Heat Charged reagent gas formed Charge transfer to analyte Analyte ions Slide 14 The LC mobile phase can be more involved in the ionization process in APCI than in ESI. ph and ionic strength tend to more important in ESI, than solvent composition or choice of organic modifier. MeOH tends to work better than acetonitrile in APCI, although ACN is satisfactory for many analytes.

15 Atmospheric Pressure PhotoIonization (APPI) APCI APPI HPLC inlet HPLC inlet Nebulizer Nebulizer Vaporizer (heater) Vaporizer (heater) Drying gas Drying gas hν Corona needle Capillary UV Lamp Capillary Slide 15 APPI is the newest member of the API family. The mode of ionization is either direct photoionization of analyte, or indirect ionization by ionized mobile phase or dopant (added to mobile phase or post-column at low levels). The corona discharge needle of APCI is replaced by a UV lamp whose wavelength has been chosen to be below the ionization potential of most LC solvents.

16 APPI Source Lamp Source instead of Discharge Needle Slide 16

17 Theory: APPI Analyte containing aerosol Evaporation hυ Photon ionizes analyte UV light Vapor hυ Dopant is photoionized and acts as reagent gas Analyte ions Slide 17

18 Comparison of Ionization Modes Mode Ionization Applicability ESI Soft Occurs in solution Basic, moderately-polar, very polar cpds APCI Thermal vap, corona discharge Extends API to less-polar cpds Requires some volatility and thermal stability APPI Thermal vap, photoionization of mobile phase or analyte Complementary to ESI and APCI Can use with dopant in mobile phase Slide 18

19 API LC/MS Techniques vs. GC/MS 1, API-Electrospray Molecular Weight 1, 1 AP PhotoIonization (APPI) APChemical Ionization (APCI) GC/MS Nonpolar Analyte Polarity very polar Slide 19

20 Some Forensic Application Examples of LC/MS LC/MS analysis of cocaine, norcocaine and benzoylecgonine in urine LC/MS analysis of opiates and metabolites in blood Rapid multi-class LC/MS drug screen Slide 2

21 Determination of Cocaine and Metabolites in Urine using LC/MS From Agilent application note, co-authors Matthew Slawson, Kimberly Shaw, Center for Human Toxicology, University of Utah, and John Hughes, Agilent Technologies

22 Introduction Cocaine and commonly-analyzed metabolites benzoylecgonine and norcocaine in urine can be analyzed using electrospray LC/MS. Analysis is done without derivatization. Sample preparation uses the same SPE as GC/MS analysis. Slide 22 Widely-abused cocaine has two metabolites commonly used as markers for cocaine usage. The typical GC/MS analysis requires derivatization of the BE metabolite, an extra complication and cost of the analysis which can be avoided by using API LC/MS. These are basic molecules which have excellent sensitivity in ESI. Samples can be analyzed using already-developed SPE sample prep, omitting the steps involved in derivatization, and reconstituting in LCcompatible solvent.

23 LC/MSD System Agilent 11 LC/MSD with patented orthogonal electrospray source Agilent 11 LC with binary pump and degasser autosampler thermostatted column compartment diode array UV/VIS detector LC/MS ChemStation for acquisition and data analysis Slide 23 A first-generation G1946A instrument was used to generate the data for this work. A G1946B and D were used in development in the Agilent laboratory. The D or SL model affords 1X increase in sensitivity and multi-signal capability for analysis of more complex samples requiring, for instance, alternating positive/negative ionization mode.

24 LC/MS Conditions Chromatographic Conditions Column: Metasil Basic 3 µm, 3 x 15 mm (Metachem) Mobile phase: A =.1% formic acid in water B= methanol Isocratic: 51% B Flow rate:.2 ml/min Column temp: 4 C Injection vol: 2 µl Diode-array detector: signal 234, 8 nm; reference 36, 1 nm MS Conditions Source: ESI Ionization mode: positive Capillary V: 15 V Nebulizer: 2 psig Drying gas : 1 L/min, 3 C SIM ions: m/z 29.1 (BE and norcocaine) m/z (BE-d3) m/z 34.1 (cocaine), m/z 37.1 (cocaine d3) Peak width:.1 min Time filter: On Fragmentor: 7 V Slide 24 The column used here was chosen because it was in use for other assays in this customer s lab. The analysis can also be carried out on Zorbax SB-C18 or Eclipse XDB-C18, and on 2mm or 4mm I.d. columns. The isobaric BE and norcocaine can be separated either by gradient or isocratic separation. The isocratic separation shown here has the productivity advantage of not requiring column re-equilibration between injections. Greater separation of the two is achieved with a gradient analysis. The lower flow rate on a small-bore LC column consumes less solvent and generates less waste than using a 4.6mm I.d. column. Resolution and MS sensitivity will also be improved with smaller i.d. columns. 3µ particle size in the column allows more rapid analysis with a shorter column having the same resolving power as a longer column with 5µ particles. Deuterated int stds were used as shown.

25 Sample preparation Clean-Screen DAU SPE columns (ZSDAU2, United Chemical Technologies) Condition with : 3 ml methanol 3 ml MilliQ water 3 ml 1mM phosphate buffer, ph6 Mix 1 ml urine with 1 ml phosphate buffer Add cocaine-d3 and BE-d3 internal standards Load on SPE column Wash column with: 2 ml MilliQ water 2 ml 1mM HCl 3 ml methanol Dry with full vacuum 5 minutes Elute with 3 ml CH 2 Cl 2 /IPA/NH 4 OH (78/2/2) Evaporate to dryness Reconstitute in 5 µl mobile phase, inject 2 µl Slide 25 A fairly standard SPE procedure designed for basic drugs works well for these analytes. Elution with the typical solvent mix for basic drugs is followed by solvent exchange into LC mobile phase for injection. Only a portion of the final sample is injected, allowing re-analysis if necessary without re-extraction of the sample.

26 Separation of cocaine and metabolitesoverlaid extracted ion chromatograms Cocaine Norcocaine Benzoylecgonine min norcocaine and BE are isobaric, thus important to be well-separated for accurate quantitation Norcocaine can also be quantitated if desired; if so, gradient separation preferred for better separation from cocaine Slide 26 These are overlaid extracted ion chromatograms for the three analytes. Note the excellent separation of the two isobaric analytes in less than 6 minutes.

27 Extracted ion chromatograms of blank urine extract m/z 29 m/z 34 No interferences found in the cocaine and BE retention windows 1 5 m/z 293 BE-d m/z 37 Cocaine-d min Slide 27 Blanks are extremely clean with this sample preparation and analysis. Note the size of the IS signals.

28 Extracted ion Chromatograms of Fortified Urine Extract m/z 29 m/z BE Cocaine BE-d3 Control urine fortified with cocaine and BE at 25 ng/ml 4 2 m/z Cocaine-d3 4 m/z min Slide 28 Typical cutoff for BE is 3 ng/ml (U.S. HHS). These levels represent less than 1/1 th the cutoff value, and still exhibit excellent S/N and large area counts for good statistics.

29 Calibration curves for cocaine and BE Cocaine, MSD1 34 BE, MSD1 29 Height Ratio Height Ratio Correlation: Correlation: Amount Ratio 2 Amount Ratio Calibration range 25-1 ng/ml Linear across calibration range without special weighting or curve treatment, r 2 >.99 Slide 29 The calibration range for this method extends to more than three times the BE cutoff, and is linear for both analytes including the origin and without special curve fitting.

30 Method Accuracy and Precision Cocaine BE Mean QC samples fortified at 5 and 15 ng/ml for cocaine and BE respectively Accuracy within 12% of target value for cocaine and 3% for BE (single validation study) GC/MS assay with same SPE is within 4% of target for cocaine, 5% for BE (previous study) CV s of 7% and 5% compare favorably with 7% for GC/MS assay StdDev C.V.* 7.1% 5.1% Slide 3

31 Additional Features of Method This single-ion SIM method has been used for toxicological, clinical and research samples on a large scale. Quantitation of norcocaine (an additional marker of cocaine use) is feasible using this method if required. This method was optimized for sensitivity by maximizing the molecular ion and minimizing CID fragmentation. Confirming (qualifier) fragment ions can be generated by CID for use with ion ratio confirmation (as in previously published LSD method). Slide 31

32 Summary: LC/MS Analysis of Cocaine and BE in urine Routine method using standard SPE sample preparation already developed for GC/MS assay LC/MS method is simpler than GC/MS method, due to no derivatization: shorter sample prep time fewer reagents less variability Overall analysis time per sample shorter than for GC/MS. Less maintenance required of LC/MSD than for GC/MS with derivatives LC/MS analysis for these analytes in urine thus offers several advantages over GC/MS, with comparable accuracy and precision. Slide 32

33 Acknowledgements Other staff of Center for Human Toxicology for GC/MS data and SPE methodology: Doug Rollins, Dennis Crouch, Dave Moody, Alan Spanbauer Christine Miller, Agilent Technologies, for review and helpful comments This information published as Agilent application note E, authors: Matthew Slawson, Kimberly Shaw Center for Human Toxicology, University of Utah John Hughes Agilent Technologies, Pleasanton, CA Slide 33

34 Determination of Opiates and Metabolites in Blood Using Electrospray LC/MS Talk derived from Agilent application note EN

35 Background Opiates are abused both as illicitly-obtained drugs and in prescription form Opiates and their metabolites are basic compounds that show excellent sensitivity in ESI Blood concentrations are normally high enough to allow use of scan mode; allows concomitant identification of other drugs in sample Slide 35 Opiates of forensic interest include heroin (with the unique marker, 6- monoacetylmorphine), and the pharmaceutical drugs shown on the next slide. All of these must be derivatized for gcms analysis, with the extra complications and efforts required for those procedures. These drugs are excellent candidates for LC/MS analysis because of their behavior as basic drugs and therefore easily protonatable. It turned out in method development, that SIM mode was unnecessary due to the relatively high concentrations of these drugs normally found in blood (up to 1ug/mL = 1mg/L). Use of scan mode allows spectra and extracted ion chromatograms to be used to locate and identify other drugs present in the sample as well.

36 Structures of Opiates and Internal Standard O CH 3CO HO HO O CH3CO O heroin (diacetylmorphine) C 21H 23NO NCH 3 O CH3CO O NCH3 6-acetylmorphine (6-monoacetylmorphine, 6-MAM) C19H21NO HO O morphine C 17H 19NO NCH 3 CH 3O HO CH 3O HO O H codeine C 18H 21NO NCH 3 O O hydromorphone C 17H 19NO NCH 3 O O hydrocodone C18H21NO NCH 3 HO CH 3O HO O nalorphine (IS) C 19H 23NO NCH 2CH CH 2 O O OH oxycodone C 18H 21NO NCH 3 Slide 36 A single non-labeled internal standard was chosen for this analysis, to allow it to be used for many analytes. Nalorphine was already in use in the Montana lab as an IS for a gcms opiates assay.

37 Instrumentation Agilent 11 LC system Vacuum degasser Binary Pump Autosampler Thermostatted column compartment DAD detector with 1.7µL microcell Agilent 11 LC/MSD VL ( standard ) model ESI source Slide 37 This method was developed and is in use on a VL model LCMSD. The SL model has approximately 1X increase in sensitivity, and can acquire multiple signals simultaneously (such as alternating positive/negative, or SIM/scan). Although the DAD is used primarily for method development, its UV spectra are acquired simultaneously with the MS data, and can also be used for drug identification and confirmation.

38 Analysis Method Chromatographic Conditions Column: Supelco Discovery HSC18, 4.6 mm x 15 cm, 3 µm Mobile phase: A =.1% formic acid in water B= methanol Gradient: start with 5%B at 2 min 5% B at 1 min 9% B at 2 min 9% B Flow rate:.5 ml/min Column temp: 5 C Injection vol: 1 µl Diode-array detector: signal 214, 8 nm; reference 36, 1 nm (used for method development only) MS Conditions Source: ESI Ionization mode: positive Vcap: 3 V Nebulizer: 4 psig Drying gas flow: 13 L/min Drying gas temp: 35 C Mass range 1-65 Fragmentor 12V Stepsize.1 Peak width:.12 min Time filter: On Slide 38 A simple mobile phase with a straightforward gradient works nicely for both the separation and MS detection. A Zorbax SB-C18 column gives equivalent performance with longer lifetime under the acidic conditions. This particular column is actually used at less than optimal flow rate to achieve a desired separation. A good separation is necessary to ensure reliable quantiation of the isobaric analyte pairs: morphine, hydromorphone m/z 286 codeine, hydrocodone m/z 3 MS parameters were optimized for maximum molecular ion production for sensitivity, and for minimized noise.

39 Sample Preparation 1 ml of blood treated with β-glucuronidase (Patella vulgata) to hydrolyze conjugates Add IntStd (1µg/mL), equilibrate, add 2mL NH 4 CO 3 ph 9, mix, centrifuge Load on conditioned UCT SPE column, elute by gravity flow, vacuum-dry Elute with 3mL MeOH and gravity flow, evaporate eluate to dryness with N2 Reconstitute in 5µL mobile phase, centrifuge Inject 1µL Slide 39 Glucuronidase used to hydrolyze any possible conjugates to parent drug. SPE procedure typical for basic drugs. Recoveries are excellent, ranging from 85-1%. Only a portion of final sample injected, allowing for re-analysis.

40 Extracted Ion Chromatograms of Opiates and IntStd MSD1 312, EIC=311.7:312.7 (OPIATES\LCMS42.D) API-ES, Pos, Scan, Frag: nalorphine-is MSD1 286, EIC=285.7:286.7 (OPIATES\LCMS42.D) API-ES, Pos, Scan, Frag: hydromorphone morphine MSD1 3, EIC=299.7:3.7 (OPIATES\LCMS42.D) API-ES, Pos, Scan, Frag: codeine hydrocodone MSD1 328, EIC=327.7:328.7 (OPIATES\LCMS42.D) API-ES, Pos, Scan, Frag: acetylmorphine min min min MSD1 298, EIC=297.7:298.7 (OPIATES\LCMS42.D) API-ES, Pos, Scan, Frag: oxycodone min min Slide 4 Analytes elute in same region of chromatogram due to similarity in structures and polarity. Morphine peakshape is better on Zorbax SB-C18 column.

41 EICs of blank blood fortified with IS MSD1 312, EIC=311.7:312.7 (OPIATES\LCMS22.D) API-ES, Pos, Scan, Frag: nalorphine-is 8 (1µg/mL) IntStd MSD1 286, EIC=285.7: (OPIATES\LCMS22.D) 6 8 API-ES, Pos, 1 Scan, Frag: min hydromorphone MSD1 3, EIC=299.7:3.7 (OPIATES\LCMS22.D) API-ES, Pos, Scan, Frag: min codeine, hydrocodone MSD1 328, EIC=327.7:328.7 (OPIATES\LCMS22.D) API-ES, Pos, Scan, Frag: 12 min MAM MSD1 298, EIC=297.7:298.7 (OPIATES\LCMS22.D) API-ES, Pos, Scan, Frag: 12 min oxycodone min Slide 41 Blanks are extremely clean- compare the Y-scale of analyte channels to that of the IS at 1ug/mL. Typical low-concentration peaks are 1 s of thousands of counts in Y-scale. Peak in the 6-MAM EIC is separated from the analyte by 1 minute.

42 Control blood fortified with analytes at.25 µg/ml MSD1 312, EIC=311.7:312.7 (OPIATES\LCMS25.D) API-ES, Pos, Scan, Frag: nalorphine-is MSD1 286, EIC=285.7:286.7 (OPIATES\LCMS25.D) API-ES, Pos, Scan, Frag: hydromorphone morphine MSD1 3, EIC=299.7:3.7 (OPIATES\LCMS25.D) API-ES, Pos, Scan, Frag: codeine hydrocodone min min MSD1 328, EIC=327.7:328.7 (OPIATES\LCMS25.D) API-ES, Pos, Scan, Frag: acetylmorphine min MSD1 298, EIC=297.7:298.7 (OPIATES\LCMS25.D) API-ES, Pos, Scan, Frag: oxycodone min min Slide 42 Signal-to-noise is very large at 25ng/mL for all analytes. Note y-axis scale at > 1,.\ Note the earlier and later portions of chromatogram in which other drugs may appear without inteference with opiates.

43 Method Accuracy and Precision Calibration range µg/ml for all analytes Typical calibration curves are linear, no special treatments, r 2 >.99 Reliable quantitation down to at least.1 µg/ml (1 ng/ml) QC samples (n=1) fortified at.25 µg/ml: CV s typically < 5% Quantitation results within 5% of target value (within 1% for 4 of 6 analytes) Slide 43 Intra-assay accuracy and precision were determined with 1 samples fortified at.25ug/ml. CV s were comparable to or better than those typically obtained with GC/MS methods, as was the accuracy. The method has been further validated and is in routine use for case work.

44 Typical Calibration Curves morphine, MSD1 286 Area Ratio = *AmtRatio Area RatioRel. Res%(1): e Correlation: Amount Ratio 5 hydromorphone, MSD1 286 codeine, MSD1 3 Area Ratio = *AmtRatio Area Ratio = *AmtRatio Area RatioRel. Res%(1): Area RatioRel. Res%(1): e Correlation: Amount Ratio Correlation: Amount Ratio oxycodone, MSD1 298 Area Ratio = *AmtRatio Area RatioRel. Res%(1): Correlation: Amount Ratio 5 hydrocodone, MSD1 3 6-acetylmorphine, MSD1 328 Area Ratio = *AmtRatio Area Ratio = *AmtRatio Area RatioRel. Res%(1): Area RatioRel. Res%(1): e Correlation: Amount Ratio Correlation: Amount Ratio Slide 44 This is not supposed to be an eye test, only to illustrate the ease of calibration with this method.

45 Method Accuracy and Precision Target concentrations.25 µg/ml morphine hydromorphone codeine hydrocodone 6mam oxycodone mean standard deviation coefficient of var percent error 1.%.36%.48% -2.16%.32% 5.16% Coefficient of var = (std dev/mean) x 1 percent error = (mean/target) x 1 Slide 45

46 Positive Blood Sample with Morphine and Hydromorphone 6 MSD1 312, EIC=311.7:312.7 (OPIATES\LCMS218.D) API-ES, Pos, Scan, Frag: nalorphine-is Decedent with opiate prescription history Morphine.84 µg/ml Hydromorphone.8 µg/ml MSD1 286, EIC=285.7:286.7 (OPIATES\LCMS218.D) API-ES, Pos, Scan, Frag: morphine hydromorphone min min Slide 46 Decedent had prescriptions for MS Contin (morphine sulfate) and Dilaudid (hydromorphone). Enzymatic hydrolysis was used for this case.

47 Positive blood sample with codeine and oxycodone Unconscious male with drug abuse history; Oxycontin found in pocket 4 MSD1 312, EIC=311.7:312.7 (OPIATES\LCMS219.D) API-ES, Pos, Scan, Frag: nalorphine-is MSD1 3, EIC=299.7:3.7 (OPIATES\LCMS219.D) API-ES, Pos, Scan, Frag: 12 min Codeine clearly present but < low calibrator (<.5 µg/ml) codeine MSD1 298, EIC=297.7:298.7 (OPIATES\LCMS219.D) API-ES, Pos, Scan, Frag: oxycodone min Oxycodone.23 µg/ml min Slide 47 Oxycontin (time-release form of oxycodone) is becoming a serious abuse problem in certain parts of the US. Codeine was clearly present well above background. Detection limits are at least.1 ug/ml.

48 Summary of LC/MS Method for Opiates in Whole Blood Method uses standard SPE sample prep without special modifications No derivatization lowers cost, sample preparation time, labor, variability Sensitivity of Agilent LC/MSD allows use of scan mode, which allows qualitative identification of other drugs in same analysis Accuracy and precision (even in scan mode!) comparable to GC/MS SIM methods Slide 48 LC/MS offers its typical advantages over GC/MS for this analysis: no derivatization, easier sample prep in other ways, larger sample volume can be injected. The LC/MSD has been demonstrated to be easy to use and robust in many forensic labs. The users at this lab maintain that every forensic tox lab should have several! This analysis is easily carried out with the VL model. The SL affords increased sensitivity for analytes with lower target concentrations, and increased flexibility for more complex analyses with its multi-signal capability. Even in scan mode, this assay on the LC/MSD gives accuracy and precision comparable to those of GC/MS SIM methods. The users have utilize a user-created MS library to identify unknown drugs using this method. The HPLC separation and MS detection also works well for benzodiazepines in blood, with a different sample preparation.

49 Acknowledgments Christine Miller, Agilent Technologies, for review and helpful comments David Presser, Agilent Technologies, for assistance in method development Dennis Crouch and David Andrenyak, Center for Human Toxicology, U of Utah, for useful insight in sample preparation This information published as Agilent Technologies application note EN, authors: Scott A. Schlueter and James D. Hutchison, Jr. Montana Department of Justice, Division of Forensic Science John M. Hughes Agilent Technologies Slide 49 Chris Miller serves as editor for many LC/MS application notes and always has helpful suggestions. David Presser also supports this customer laboratory and did much of the initial user training. The staff of the Center for Human Toxicology generously shares its expertise in sample preparation with many tox labs world-wide. The Center also uses Agilent GC/MS and LC/MS.

50 Combined Screening and Confirmation of Drugs of Abuse in Postmortem Specimens Using LC-MS Following Solid-phase Extraction

51 Method Development Rationale Objective: develop a broad-spectrum drugs-of-abuse sample preparation, screening and confirmation method using LC-MS analysis Based on - SPE method [Bogusz et al, JAT, 24:77-84 (2)] - LC-MS method for serotonergic drugs [Goeringer et al, JAT, 27:3-35 (23)] Slide 51

52 Sample Preparation/Extraction (Bogusz et al, JAT 2) Tissue sample (5 µl) IS (1 µl) Centrifuge 5 x g Supernatant (5 µl) ph 9.3 Ammonium Carbonate Buffer (2 ml) Vortex, centrif 1 5 x g 2-mg Bond Elute C 18 cartridges Condition: Methanol (1 ml) Water (1 ml) ph 9.3 Ammonium Carbonate Buffer (2 ml)inject Supernatant applied to SPE cartridges Wash SPE cartridge: ph 9.3 AmmCarb buffer (2mL) Vacuum dry 5 min Elute sample: 9:1 methanol/.5m acetic acid (.5 ml) Add 1. mmol HCl (1 µl) Centrifuge 5 14, x g Evaporate, reconstitute in mobile phase, inject 3µL Slide 52

53 LC/MS Conditions Column: Zorbax Extend-C 18 (2.1 x 15 mm, 5 µm particle size, Agilent Technologies Pty Ltd) Mobile phase: 55/44.5/.5 NH 4 OH/MeOH/THF, ph C, 16 run Agilent 11 Series HPLC with G1946A MSD Electrospray ionization in positive mode MS Conditions: Frag voltage Neb press Cap voltage Drying gas flow rate Drying gas temp 8 V 3 psig 35 V 1 L/min 325 deg C Slide 53

54 SIM ions for some target analytes (quant ions underlined) Compound Ions Monitored Amphetamine 119, 12, 136 BZE 29, 291, 292 Flunitrazepam 314, 315, 316 MDMA 194, 195, 196 Methamphetamine 119, 12, 15 Morphine 286, 287, 289 Slide 54

55 LC-MS Separation of Drugs of Abuse benzoylecgonine amphetamine IntStd morphine flunitrazepam Slide 55

56 Comparison of Results to Current Methodology (GC/MS) Results from 9 cases also analyzed using GC/MS (GC/MS results shown in parentheses). Case # Amphetamine BZE Morphine Oxazepam 1.15 (.12) (.8).5 (ND) (.49) (.9) (1.19) (.1) (.5) (.17) (.3) Slide 56

57 Detectable Drugs Using Current Method Method has been applied to analysis of 45 drugs and metabolites: Drug RT Drug RT Drug RT cotinine.53 moclobemide 8.84 nordazepam aminonitrazepam 1.47 pholcodine 8.92 quinine sulfate aminoclonazepam 1.51 strychnine 9.54 citalopram aminoflunitrazepam 2.27 flunitrazepam 9.69 sertraline 5.97 BZE 2.62 prochlorperazine diazepam phentermine 3.2 nitrazepam desipramine 58.1 MDMA 3.38 chlorpromazine olanzapine 63.2 morphine 3.61 fenfluramine 2.19 paroxetine pseudoephedrine 4.11 triazolam benztropine 8.62 trifluoperazine 5.94 temazepam ephedrine amphetamine 6.66 propranolol bupivicaine codeine 6.72 venlafaxine 29.4 haloperidol methamphetamine 7.56 cocaine 3.96 doxepin hydrocodone 7.89 nortriptyline 31.1 promethazine oxycodone 7.99 chlorpheniramine mianserin Slide 57

58 Summary- Drug Screen by LC/MS Extraction/SPE procedure recovers a wide variety of drugs from post-mortem matrices. Useful for determination of all drugs of abuse except THC or carboxy-thc Method has been used to analyze blood, brain, urine, vitreous fluid, & bile in 3 death investigations Use of newer LC/MSD s would provide increased sensitivity; with LC/MSD Trap, MS/MS capability Slide 58

59 Acknowledgements Authors: Kabrena E. Goeringer, Iain M. McIntyre, and Olaf H. Drummer, Victorian Institute of Forensic Medicine, Monash University, Australia The authors are grateful to the staff of the Victorian Institute of Forensic Medicine for their assistance with tissue collection, and to the next of kin who kindly gave permission to use their family members tissues for research purposes. This information was presented by Dr. Goeringer at SOFT 21, and published in JAT 27:3-35 (23). We thank Dr. Goeringer for sharing the presentation and Prof. Drummer for his kind permission to reproduce some of the information. Slide 59

60 Points to Take Away Wide applicability of API LC/MS Small to large molecules, gc able or not No derivatization, simplified sample preparation LC/MSD - Routine, robust, quantitative Orthogonal Spraying Automatic tuning and calibration Low maintenance API Electrospray - Spray then evaporate Soft ionization, high sensitivity, optimize ionization mode APCI Vaporize then ionize Requires some thermal stability of analyte APPI Complimentary to ESI and APPI Most like APCI (heated vaporization) Slide 6

61 E-Seminar questions? or (alien visitor to local tox lab, properly wearing visitor s badge) Slide 61