Two-Dimensal LC/MS/MS to Reduce Ion Suppress in the Determinat of Cannabinoids in Blood Plasma Applicat Note Forensic Toxicology Authors Gerd Vanhoenacker, Mieke Steenbeke, Koen Sandra, and Pat Sandra earch Institute for Chromatography President Kennedypark B-8 Kortrijk Belgium Udo Huber and Sonja Schneider Agilent Technologies, Inc. Waldbronn, Germany Abstract Cannabinoids in plasma were analyzed after minimal sample pretreatment with an Agilent 9 Triple Quadrupole LC/MS. Significant loss of signal compared to analysis of standard soluts was observed in plasma samples. Matrix-related suppress could be overcome by using the Agilent 9 Infinity II D-LC solut with high-resolut sampling. The setup was tuned to enable simultaneous analysis of the psychoactive THC (Δ-9 tetrahydrocannabinol) and metabolites carboxy-thc () and hydroxyl-thc (THC-OH) in blood plasma.
Introduct The analysis of cannabinoids in biological matrices such as saliva, urine, hair, and blood is of utmost importance in forensic toxicology laboratories. Cannabis, or marijuana, is one of the most frequently used illegal drug substances. Detect of Δ-9 tetrahydrocannabinol (THC) or metabolites in plasma is indicative of recent usage, and can can be benificial in driving under-the-influence or professal screening campaigns,. THC is the principal psychoactive component of cannabis, and is quickly metabolized and cleared from blood. The main metabolites are carboxy THC (THC COOH) and hydroxyl THC (THC OH). Both can be detected in biological matrices over a longer period. Detect of THC in plasma indicates recent use, whereas detect of metabolites can be an indicat of past use. Analysis of these substances in whole blood and plasma often requires extensive sample preparat to reduce the amount, or remove proteins, lipids and phospholipids, and other potential interfering compounds. Therefore, protein precipitat, liquid-liquid extract, solid-phase-extract, and their combinats are needed prior to analysis with LC/MS/MS. Sample preparat is a time-consuming and exhaustive part of the analytical scheme, and ways to bypass this are sought after. The main problem for biological matrices such as blood plasma, is that a substantial amount of matrix material enters the izat source and influences the izat process. In most cases, this results in reduced analytical sensitivity, known as suppress. Analytical sensitivity is also reduced in the long term due to fouling of the source and the MS entrance. Performing a heart-cut on the chromatographic reg of interest and analyzing the cut(s) on a second dimens column with orthogonal selectivity can strongly reduce or even eliminate izat suppress. Using the Agilent 9 Infinity II D-LC solut to transfer small fracts of a first-dimens separat, in which the THC signal was nearly completely suppressed by plasma matrix constituents, onto a second dimens, reduced izat suppress to such an extent that accurate quantificat of THC together with THC-OH and in blood plasma was obtained after minimal sample pretreatment. Experimental Chemicals, samples, and sample preparat Acetonitrile was HPLC-S quality, and water and methanol were ULC/MS quality. Solvents were purchased from Biosolve (Valkenswaard, The Netherlands) Ammonium acetate and formic acid were from Sigma-Aldrich (Bornem, Belgium). Stock soluts in methanol of THC, THC COOH, and THC-OH. Their respective labeled internal standards THC-d, THC COOH-d, and THC-OH-d were from Sigma-Aldrich (Bornem, Belgium). These were mixed and diluted to the appropriate concentrat ( to ng/ml for standards, and ng/ml for the internal standards) in. % formic acid in acetonitrile. A port of this solut was then mixed with an equal port of water to obtain the standard for inject in water/acetonitrile containing. % formic acid and ng/ml of the internal standards. Plasma samples were prepared by mixing µl plasma with µl of. % formic acid in acetonitrile containing ng/ml of the internal standards (and standards for spiking experiments). The acetonitrile phase was added in four µl aliquots, and after each addit, the sample was vortexed for seconds. The complete mixture was then centrifuged for minutes at, g. The clear upper phase was transferred to an autosampler vial, and injected for analysis. Instrumentat An Agilent 9 Infinity II D-LC solut was used. The configurat is described below. LC/MS configurat for one dimensal analyses Agilent 9 Infinity II High Speed Pump (G7A) Agilent 9 Infinity II Multisampler (G77B) with µl analytical head and µl loop Agilent 9 Infinity II Multicolumn Thermostat (G7B) with -posit/-port valve head (GB) and divider assembly (G7-) Agilent low dispers kit for Agilent 9 Infinity II LC (7 9) Agilent 9 Triple Quadrupole LC/MS (G9A) with Jet Stream technology source Addital modules for two dimensal analyses Agilent 9 Infinity II High Speed Pump (G7A) Agilent 9 Infinity Valve Drives (x G7A) with D-LC valve (-posit/-port duo-valve, GA) and multiple heart-cutting valves (x G-) equipped with µl loops Figure shows the configurat of the valves for multiple heart-cutting with the Agilent 9 Infinity II D-LC solut. Software Agilent OpenLab CDS ChemStat Edit software, vers C..7 [7] with Agilent 9 Infinity D-LC acquisit software, vers A.. [] was used for control of the D-LC system, and Agilent MassHunter acquisit software B.8. for the triple quadrupole LC/MS system.
IN Waste ¹D-Column D-Pump Autosampler D-Column OUT 8 LC/MS IN OUT Figure. Instrument and valve configurat for multiple heart-cutting analysis. 7 ²D-Pump ²D-Column
Method for one-dimensal and first-dimens analysis Parameter Value Column Agilent ZORBAX Eclipse Plus C8 RRHD,. mm,.8 µm (p/n 9977-9) Prefilter Agilent 9 Infinity inline filter,. µm, (p/n 7-8) Mobile phase A Mobile phase B Flow rate mm Ammonium acetate in water Methanol. ml/min Gradient to minute: %B to 9 minutes: to %B Column temperature Left, C For two-dimensal analysis, the valve was switched from Posit to Posit after:. minutes ().8 minutes (THC-OH) 7. minutes (THC) Inject Injector temperature 8 C Inject program with needle wash (flush port, seconds, methanol) Draw µl sample Draw µl. % formic acid in water Inject Second-dimens analysis with high-resolut sampling Parameter Column Mobile phase A Mobile phase B Flow rate Value Agilent ZORBAX Eclipse Plus Phenyl-Hexyl RRHT,. mm,.8 µm, (p/n 999-). % formic acid in water Methanol. ml/min (idle flow. ml/min) Gradient to. minutes: to %B. to.9 minutes: %B.9 to. minutes: %B Cycle time. minutes D Sampling seconds (7 % loop filling) Column temperature Right, C MS source parameters Parameter Value Ionizat Agilent Jet Stream technology source Drying gas temperature C Drying gas flow L/min Nebulizer pressure psig Sheath gas temperature 8 C Sheath gas flow L/min Capillary voltage,/, V (±) Nozzle voltage /,7 V (±) Ion funnel high-pressure RF /9 V (±) Ion funnel low-pressure RF / V (±)
MS acquisit parameters Segment : to. minutes, to waste Segment ():. to.9 minutes, to MS Compound Precursor MS Product MS Dwell* (ms) Collis energy (V) Cell accelerator voltage (V) Polarity. Unit 99. Unit /. Negative. Unit. Unit / Negative. Unit 9. Unit / Negative d. Unit 8. Unit / Negative d. Unit. Unit / Negative Segment (THC-OH):.9 to 7. minutes, to MS Compound Precursor MS Product MS Dwell (ms) Collis energy (V) Cell accelerator voltage (V) Polarity THC-OH. Unit. Unit /. Positive THC-OH. Unit. Unit / Positive THC-OH. Unit 9. Unit / Positive THC-OH d. Unit 9. Unit / Positive THC-OH d. Unit. Unit / Positive Segment (THC): 7. minutes, to MS Compound Precursor MS Product MS Dwell (ms) Collis energy (V) Cell accelerator voltage (V) Polarity THC. Unit 9. Unit / Positive THC. Unit 9. Unit / Positive THC. Unit Unit / Positive THC d 8. Unit Unit / Positive THC d 8. Unit 9. Unit / Positive *Dwell time was set at ms for D acquisit, at ms for D acquisit.
ults and Discuss One-dimensal analysis Because the extracts contain approximately % acetonitrile, and relatively large volumes are injected, an inject program was mandatory to focus the analytes on the column. THC COOH particularly suffers from excessive band broadening and peak splitting when injected in an overly strong solvent. Injecting a µl plug of. % formic acid in water helps improve the peak shape by diluting the acetonitrile, and neutralizing. As a result, inject of µl of the sample was made possible without significant peak broadening. Following the inject of a standard solut, the three cannabinoids were well separated and clearly detected. When injecting a spiked plasma extract, some interferences and izat suppress hinder the analysis. Figure shows examples of such analyses. The inject was first carried out on a mm long column. In this case, the signal for THC was completely suppressed, and the signal for THC-OH showed approximately 7 % suppress (based on comparison of peak areas). One way to reduce izat suppress is to increase the chromatographic resolut. The most obvious way to do this is to use a longer column. A -mm column was installed, and the analysis of standard and sample was repeated. The bottom part of Figure shows that this reduced the suppress for the THC-OH peak from 7 to 8 %, and that a small signal was detected for THC. However, the response for THC in the plasma was too small to be used for quantitative purposes. As THC-OH and are detected on both columns, this strategy can be applied to quantify them by isotope dilut after addit of labeled internal standards. Analysis of a nonspiked plasma sample revealed the presence of and THC-OH, and indicated that the donor had used cannabis..8 A.....8.....8.......9.8.7...... (Present in plasma sample)..8.....8.....8 7. 7. 7. 7. 7.8 8. 8. 8. 8. 8.8 9. 9. 9. 9. 9.8. Acquisit time (min) B (Present in plasma sample) THC-OH 7 % suppress THC-OH 8 % suppress THC Complete suppress D mm column Standard ng/ml Plasma spiked with ng/ml D mm column Standard ng/ml Plasma spiked with ng/ml THC Severe suppress..8.....8.....8 7. 7. 7. 7. 7.8 8. 8. 8. 8. 8.8 9. 9. 9. 9. 9.8. Acquisit time (min) Figure. Total current for the D analysis of a standard solut and plasma extract (both soluts contain ng/ml of each analyte and no internal standards) on a mm (A) and mm (B) column. The gradient was identical for both analyses (see Experimental sect).
A solut for the loss in signal for the THC peak can be found in an exhaustive sample preparat step to purify the extract further and fractate THC better. In this case, it was likely that izat suppress was caused by the phospholipids present in the plasma. Another opt is to collect the peak (or elut window) of interest, and reanalyze this fract on a second column/mobile phase combinat with different selectivity compared to the original analysis. This could separate the target from the abundant matrix component(s) and reduce or even eliminate the suppress. 8 7 A Positive izat 7 cuts over entire peak Standard ng/ml Plasma sample Cut Cut Cut Cut Cut Flush Cut 7 Cut Impurities Impurities Two-dimensal analysis Offline collecting and reinjecting fracts is time-consuming, and there is a considerable risk of sample loss or contaminat in intermediate steps (for example, drying and redissolving). The procedure can be performed online with the Agilent 9 Infinity II D-LC solut and MHC opt. The THC peak can be sampled in small fracts, and each of these fracts can then be analyzed automatically on a second-dimens column. In this study, a phenyl-hexyl statary phase with a formic acid and methanol mobile phase was selected. Although and THC-OH could be analyzed in the D-LC analyses, some D-LC tests were carried out to have a better understanding of the impact of sample matrix on these compounds as well. High-resolut sampling on the peak was done with positive and negative izat. In positive izat, significant amounts of interferences with the same transit but different ratios were detected. These were separated from the in the second dimens due to the orthogonal separat behavior, as shown in Figure. 7 8 9 7 8 9 Acquisit time (min) B Positive izat Cut Cut 7 m/z. 7. m/z. 99. m/z. 9. Impurities Impurities Figure. Positive izat MHC analysis of of a standard solut ( ng/ml of each analyte and internal standards) and nonspiked plasma extract. A) Total current of the complete analysis of the seven heart-cuts. B) transits in detail of Cut (contains mainly ) and Cut 7 (contains mainly the interference). 7
The relative abundance of the second dimens peaks changes between different fracts or cuts. This indicates that the peak detected in the first dimens using positive izat is not corresponding to one product. The interference is not detected with negative izat, and as is acidic, negative izat gives excellent analytical sensitivity for this compound on the 9A Triple Quadrupole MS, and was consequently selected as polar for D-LC analysis of this compound. The MHC method also confirmed the presence of in the nonspiked plasma sample. The same approach was applied to the THC-OH peak, where some suppress was observed in D-LC analyses. No significant interferences were detected with MHC, but signal suppress was reduced - to -fold (from 7 to only 8 %) in comparison with the original separat on the -mm column.. A THC-OH Standard ng/ml ( D-LC) Plasma spiked with ng/ml...8.....8.....8... ( D-LC) Cut THC ( D-LC) Cut Cut Cut Cut 7 8 9 7 8 9 Acquisit time (min) B Flush The main problem in the one-dimensal separat of samples was the (nearly) complete suppress of the signal for THC. The applicat of high-resolut sampling on this peak resulted in radical improvement of the signal. Not all signal suppress was removed, but further optimizat of second-dimens statary and mobile phase could be done to improve this. The recovery was satisfactory, and the detected peaks can be used to quantify the THC in plasma extracts. Figure shows an example of such an analysis and an overlay of Cut from five consecutive injects of a spiked plasma sample. C Cut.7.8.9 7. Acquisit time (min) Cut Cut It is clear that the procedure is repeatable, and the method was evaluated for repeatability, linearity, and accuracy. A set of standard soluts, sample, and spiked samples was prepared and injected with the final method. Single injects of standard soluts with concentrat ranging from. to ng/ml were used to evaluate linearity, and the calibrat curve was used to determine the concentrat of the analytes in the nonspiked plasma sample. Figure. MHC analysis of a standard solut and spiked plasma extract (both soluts contain ng/ml of each analyte and internal standards). A) Total current of the complete analysis ( and THC-OH in D-LC, THC in D-LC). Five cuts were taken. B) TIC overlay for Cut from five consecutive injects of spiked plasma. C) Detail of TIC for Cuts to. 8
Spiked samples were prepared at the same concentrats as the standard soluts to calculate the accuracy of the method, and inject precis was determined by five consecutive injects of both standard soluts and spiked samples ( ng/ml). Detect limits in standard soluts were below. ng/ml for all compounds. Figure shows an example of low-level standard soluts and plasma sample, and Table summarizes the main results for method evaluat. Conclus The Agilent 9 Infinity II D-LC solut with the Agilent 9 Triple Quadrupole LC/MS enables simultaneous quantificat of THC,, and THC-OH in plasma samples after minimal sample preparat. THC COOH and THC OH were analyzed after one dimensal chromatography, while THC was only detected in plasma samples after high-resolut sampling to reduce izat suppress caused by the matrix. The developed method showed good linearity and precis. The method can be highly useful to quantify compounds in difficult matrices originating from various fields. References. Hädener, M.; et al. Anal. Bioanal. Chem., 8, 9 9.. Desrosiers, N.; et al. J. Anal. Toxicol., 9,.. Crifasi, J.; Honnold, R.; Kubas, R. Agilent Technologies Applicat Note, publicat number 99-9EN,.....9.8.7........9.8.7...... Cut Standard solut. ng/ml Plasma spiked with. ng/ml Plasma sample Blank solut.......7.7.8.8.9.9 7. 7. 7. 7. 7. Acquisit time (min) Figure. Quantifier transit for analysis of Cut of THC peak in blank solut,. ng/ml standard solut, plasma extract and spiked plasma extract (. ng/ml). Table. Method evaluat results. Standards Area RSD% (n =, R² a ng/ml) Area RSD% (n =, ng/ml) Concentrat (ng/ml) Plasma Concentrat spike ng/ml (Recovery%) Concentrat spike ng/ml (Recovery%) D.997...7.7 (.).9 (.) THC-OH.99.9.9.8. (8.) 9.8 (9.) D THC Cut.99.78 7.8 THC Cut.997.9 8. THC Cut.99. 9. THC Cut. b.78 b. c. c (.8) 9.8 c (9.) ª Calibrat levels:.,.,.,,,,,, and ng/ml b RSD% calculated on the sum of the peak areas for Cuts c Average concentrat from Cuts. Van Damme; et al. Anal. Bioanal. Chem.,, 7. 9
www.agilent.com/chem For Forensic Use. This informat is subject to change without notice. Agilent Technologies, Inc., 7 Published in the USA, March, 7 99-789EN