An LC-MS-TOF Method for Quantifying Components of Interest in … · 2016. 10. 13. · Abstract. AOAC 2016. Poster # PW-033. Results and Discussion. Compounds. Results and Discussion

2 x10 0 0.2 0.4 0.6 0.8 1 * 4.30817017246.00 2 x10 0 0.2 0.4 0.6 0.8 1 4.656 2 x10 0 0.2 0.4 0.6 0.8 1 * 4.526 2 x10 0 0.2 0.4 0.6 0.8 1 5.269 2 x10 0 0.2 0.4 0.6 0.8 1 * 5.922 08 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 5 52 54 56 58 6 62 64 66 68 7 72 74 76 78 8 82 84 86 88 9 92 94 96 98 10 10 2 10 4 10 6 10 8 11 11 2 11 4 11 6 11 8 CBN - 5 Levels, 4 Levels Used, 5 Points, 4 Points Used, 0 QCs Concentration (pp 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Responses 3 x10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 y = 2.626755 * x - 10.790923R^2 = 0.99720316Type:Linear, Origin:Include, Weight:None 1 x10 -0.2 0 0.2 0.4 0.6 0.8 1 +ESI EIC(315.2319) Scan Frag=175.0V THC 125 ppb.d * 5.91071132.12 1 x10 -0.2 0 0.2 0.4 0.6 0.8 1 +ESI EIC(315.2319) Scan Frag=175.0V CBD 100 ppm.d * 4.30817017246.00 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 Results and Discussion Abstract AOAC 2016 Poster # PW-033 Results and Discussion Compounds Results and Discussion Conclusions Mike Adams 1 , A. Roth 1 , Joan Stevens 2 , Karen Kaikaris 3 , & Sue D’Antonio 3 1 CWC LABS, Agilent Technologies, 2 Wilimington, DE., 3 Cedar Creek, TX. Introduction Experimental HPLC Conditions Agilent 1290 Infinity II UHPLC series Quaternary Pump, Multisampler with wash , Multi Column Themostat, DAD Column: Zorbax Eclipse Plus, C8 2.1 x 50mm, 1.8 µm Column temperature: 50°C Injection volume: 0.25 µL Autosampler temp: 23 ºC Needle wash: 3.5 s Flush Port (25:25:50) (H 2 O:IPA:MeOH) DAD-UV 254 nm Mobile phase: A = Water B= Methanol C= 0.1% Formic Acid Water Flow rate: 0.3 mL/min Gradient: Time (min) %B %C 0.0 60 5 12.0 95 5 Stop time: 15.0 min. Post time: 5.0 min. Overall run time 20.0 minutes (incl. re-equilibration) Note the sample prep consists dilution of the CBD oil. This requires a longer time for the oil to elute from the column. Allowing a longer run time ensures reproducible retention times and lower carryover. MS : Agilent 6230B Time of Flight Mass Spectrometer MS Parameters Ion Mode Dual ESI, PositiveMass Range m/z 100-1700 Scan Rate (spectra/sec) 1.00 Source Parameters Drying gas (Nitrogen) 12 L/min Drying gas temperature 350 ºC Nebulizer gas (Nitrogen) 40 PSI Scan Source Parameters Capillary Voltage 4000 V Fragmentor 175 V Skimmer 65 Ref Mass Enabled Ref Masses 121.0508730 922.0097980 Average scan 1 Detection window 100 ppm Min Height 1000 LC UV Calibration Curves* *Note that CBG was not analyzed via LC UV 1 x10 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 +ESI EIC(315.2319) Scan Frag=175.0V CBD 100 ppm.d * 5.91071132.12 * 4.30817017246.00 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 9 delta THC - 5 Levels, 5 Levels Used, 5 Points, 5 Points Used, 0 QCs Concentration (pp -50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 Responses 5 x10 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 y = 629.460195 * x - 12665.572874R^2 = 0.99910358Type:Linear, Origin:Ignore, Weight:None An LC - MS - TOF Method for Quantifying Components of Interest in Hemp Extract THC CBG CBDA CBD CBN Time of Flight Extracted Ion Chromatograms of Standards UV Chromatograms of Standards Compounds Formula M/z m/z + Na RT(Minutes) Abreviation Notes Cannabidiolic acid C22H30O4 359.2217 381.2036 4.660 CBDA Cannabinol C21H26O2 311.2006 333.1825 5.261 CBN Cannabidiol C21H30O2 315.2319 337.2319 4.308 CBD Isobaric Tetrahydrocannabinol C21H30O2 315.2319 337.2138 5.922 THC Isobaric Cannabigerol C21H32O2 317.2475 339.2295 4.530 CBG Chromatography -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 * 3.69853.00 * 5.34370643007.42 4.372518.46 3.925271.65 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5 Lowest concentration of THC Highest concentration of CBD Example (CBD) Detectors UV vs LC TOF UV Cost effective Good linearity LC TOF Good linearity Low LOQ Low LOD Compound specificity Extended dynamic range (see below) CBD - 7 Levels, 7 Levels Used, 7 Points, 7 Points Used, 0 QCs Concentration (pp -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 Responses 7 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 y = -1039.478492 * x ^ 2 + 265938.598953 * x + 41631.182274R^2 = 0.99777691Type:Quadratic, Origin:Include, Weight:1/x CBD - 5 Levels, 4 Levels Used, 5 Points, 4 Points Used, 0 QCs Concentration (pp 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 Responses 2 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 y = 0.265107 * x - 0.422537R^2 = 0.99845528Type:Linear, Origin:Include, Weight:None CBDA - 5 Levels, 4 Levels Used, 5 Points, 4 Points Used, 0 QCs Concentration (pp 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 Responses 2 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 y = 0.265107 * x - 0.422537R^2 = 0.99845528Type:Linear, Origin:Include, Weight:None LC UV Calibration Curves LC MS Calibration Curves Delta 9 THC - 2 Levels, 2 Levels Used, 2 Points, 2 Points Used, 0 QCs Concentration (pp 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 Responses -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 y = 0.734238 * x - 0.026467R^2 = 0.99762637Type:Linear, Origin:Include, Weight:None CBDA - 7 Levels, 7 Levels Used, 7 Points, 7 Points Used, 0 QCs Concentration (pp -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Responses 5 x10 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 y = 7593.366507 * x + 3848.053414R^2 = 0.99698334Type:Linear, Origin:Ignore, Weight:None 2 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 +ESI EIC(315.2319) Scan Frag=175.0V THC 12.5 ppb.d Noise (PeakToPeak) = 803.09; SNR (5.909min) = 2.3 * 5.922 * 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7 2 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 +ESI EIC(359.2217) Scan Frag=175.0V CBDA 1 ppm.d Noise (PeakToPeak) = 645.45; SNR (4.639min) = 2.9 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 2 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 +ESI EIC(315.2319) Scan Frag=175.0V CBD 1 ppm.d Smooth Noise (PeakToPeak) = 197.93; SNR (4.349min) = 212.3 * 3 4 5 6 7 8 9 Compound_4 - 7 Levels, 7 Levels Used, 7 Points, 7 Points Used, 0 QCs Concentration (pp -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Responses 7 x10 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 y = -930.600932 * x ^ 2 + 424824.440303 * x + 232588.569605R^2 = 0.99922237Type:Quadratic, Origin:Ignore, Weight:1/x 2 x10 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 +ESI EIC(311.2006) Scan Frag=175.0V CBN 1 ppm.d Noise (PeakToPeak) = 707.30; SNR (5.278min) = 200.7 5.269 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 CBG - 5 Levels, 5 Levels Used, 5 Points, 5 Points Used, 0 QCs Concentration (ng -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Responses 7 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 y = 245201.757323 * x + 99717.264587R^2 = 0.99919784Type:Linear, Origin:Include, Weight:None 2 x10 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 +ESI EIC(317.2475) Scan Frag=175.0V CBG 80 ppm.d 1 1 2 3 4 5 6 7 8 9 10 11 12 13 1 Hemp extracts contain high concentrations of cannabidiol (CBD) and low concentrations of Δ- 9-THC, the major psychoactive component of the cannabis plant from which CBD is extracted. Other compounds found in hemp oil include cannabinolic acid (CBNA), cannabinol (CBN), cannabigerol (CBG) and cannabidiolic acid (CBDA). While liquid chromatography or GC-MS have traditionally been the method of choice for analysis of hemp extract, the utilization of an LC – MS - TOF allows for a single, more accurate method of quantifying all of these hemp extract components. The method presented here allows for the quantitation of high concentrations of CBD and related components as well as the relative low concentrations of Δ-9-THC within a single analysis. Analytical run times are 20 minutes. This run time is compensated for by shorter and more accurate sample preparation. Standards are diluted automatically with the use of an autosampler. Shorter run times (<10 min) may be achieved with more extensive sample preparation, but quantitation may be less accurate. The use of a custom Personal Compound Database and Library (PDCL) allows the analyst to verify identity data by matching accurate mass and retention times and avoid the errors that come with co-elution of analytes. Additional screening for toxic contaminants can be accomplished by comparison of the data to a PCDL. Hemp extracts have recently become an area of scientific interest. Some of these extracts include Cannabidiol (CBD), Cannabidiolic Acid (CDBA), Cannabinol (CBN), Cannabigernol (CBG), and Δ-9-Tetrahydrocannabinol (Δ-9-THC). CBD, CBDA, CBN, and CBG extracts occur in relatively high to moderate concentrations and are non- psychoactive. Δ-9-THC is psychoactive but occurs in very low concentrations (<0.3%). Traditional methods for the analysis of major cannabinoids in hemp include Thin Layer Chromatography, Gas Chromatography – Flame Ionization Detection, Gas Chromatography – Mass Spectrometry, and HPLC-UV. Unfortunately, none of these traditional methods offer the dynamic range necessary to analyze the major cannabinoid components of hemp in a single analytical run. Some methods also degrade CBDA, yielding results which do not accurately represent the composition of the sample. Furthermore, some of these methods require extensive sample preparation which can result in the loss of time and analyte. The method presented here utilizes an HPLC to separate the compounds before being detected and quantified by a time-of flight mass spectrometer. One advantage to this method is that sample preparation, and therefore the risk of loss of analyte, is minimal. In addition, limits of quantification and detection are many orders of magnitude lower than those of traditional methods. Analyte retention times are confirmed with MS-MS accurate mass confirmation. Lastly, the large dynamic range of this LC-MS-TOF methods enables the analyst to quantify all the cannabinoids of interest in a single analytical run. In these experiments, Cerilliant standards C-045 (CBN), C-144 (CBDA), C-141 (CBG), C-046 (CBD) were used. For THC analysis the Agilent Forensic tox mix, 5190-047, was used. -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 +ESI EIC(315.2319) Scan Frag=175.0V blank after cbd.d * Counts (%) vs. Acquisition Time (min) 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 Carryover check blank after highest concentration of CBD Overlay of blank and 100 ppm standard 2 x10 0 0.2 0.4 0.6 0.8 +ESI EIC(315.2319) Scan Frag=175.0V CBD 100 ppm.d * 1 1 2 x10 0 0.2 0.4 0.6 0.8 +ESI EIC(315.2319) Scan Frag=175.0V blank after cbd.d 1 1 Counts (%) vs. Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 Hemp extracts contain several cannabinoids of interest. Cannabidiol (CBD) is found in relatively high concentrations while Δ-9-tetrahydrocannabinol (THC) is found in very low concentrations. This preliminary method achieves good chromatographic separation of the five cannabinoids studied and has the advantage of minimal sample preparation. CBD and Δ-9-THC may coexist in hemp extracts at four or more orders of magnitude differences in concentrations. In contrast to traditional methods which lack the dynamic range of LC-MS-TOF, this method offers a wide dynamic range of detection that simultaneously quantitates both high and low concentration cannabinoids in a single run, all in liquid form. Methods employing HPLC-UV are cost effective and exhibit good linearity, but lack the dynamic range required to detect both ends of the concentration spectrum. The utilization of this LC-MS-TOF method exhibits good linearity but also offers excellent dynamic range and has the added benefit of MS-MS confirmation of peaks. The use of this method results in limits of detection and quantitation that are orders of magnitude lower than LC-UV methods permit. 2 x10 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 * 4.308 4.530 4.660 5.261 5.922 Counts (%) vs. Acquisition Time (min) 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2 Overlay of 5 standards at same concentration 2 x10 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 * 4.306 Counts (%) vs. Acquisition Time (min) 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 4.306 Counts vs. Acquisition Time (min) 3.35 3.4 3.45 3.5 3.55 3.6 3.65 3.7 3.75 3.8 3.85 3.9 3.95 4 4.05 4.1 4.15 4.2 4.25 4.3 4.35 4.4 4.45 4.5 4.55 4.6 4.65 4.7 4.75 4.8 4.85 4.9 4.95 5 5.05 5.1 5.15 5.2 5.25 6 x10 0 0.2 0.4 0.6 0.8 1 1.2 315.2297([C21H30O2]+H)+ 337.2112([C21H30O2]+Na)+ 6 x10 0 0.2 0.4 0.6 0.8 1 1.2 315.2297(M+H)+ 337.2112(M+Na)+ Counts vs. Mass-to-Charge (m/z) 305 310 315 320 325 330 335 340 345 350 355 360 365 Extracted Ions overlaid on TIC (black) chromatogram PCDL Search Results Using the Find by Formula function with our custom PCDL, we confirmed the presence of CBD and absense of THC in this sample. + EIC (315.2309) Scan cbd 250 5000 to 1.d Acquisition Time (m 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 Counts 6 x10 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 *4.306 min. + Scan (4.215-4.365 min, 9 scans) cbd 250 10000 to 1.d Mass-to-Charge (m 314.5 315 315.5 316 316.5 317 317.5 318 318.5 Counts 6 x10 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 315.2316 316.2347 317.2369 Qualitative Analysis of CBD in Oil Quantitative Analysis of CBD in Oil CBD extracted Ion in Orange CBD CBG CBN CBDA THC Agilent products and solutions are intended to be used for cannabis quality control and safety testing in laboratories where such use is permitted under applicable state law.

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An LC-MS-TOF Method for Quantifying Components of Interest in … · 2016. 10. 13. · Abstract. AOAC 2016. Poster # PW-033. Results and Discussion. Compounds. Results and Discussion