Advancing chromatographic data quality to make a safer Cannabis
Recent Improvements in Chromatography: Advancing Chromatographic Data Quality to Make a Safer Cannabis Product Rick Lake, Jack Cochran, Amanda Rigdon, Frances Carroll, Corby Hilliard Restek Corpora,on, Bellefonte PA Outline • Review current and possible future regulaFons • Define some guidelines we can use for developing reliable chromatographic data • Define some recent chromatographic technologies that could help with future regulaFons and economics • Goal is to define an approach to cannabis methods that will fit various regulaFons, if and when they apply What will happen to Cannabis tes3ng? WHO AM I? LEGAL? ILLEGAL? INGREDIENT? FOOD? DRUG? SUPPLEMENT? 3 / 9 Regula3ons Federal Oversight Federal LegalizaFon State RegulaFons Code of CO RegulaFons Retail Marijuana Code Colorado Marijuana Enforcement Division (MED) Compassionate Access, Research Expansion, and Respect States (CARERS) Act Forms of Analy>cal Methodologies: • Provided, Promulgated methods • EPA methods • Compendial, “Choose to Use” Methods • USP • ASTM • AOAC • Independent, Validated Methods • FDA • CLIAA 4 / 9 Has a precedent been set? Ginseng Dietary Supplements Health and Educa3on Act of 1994 (DSHEA) • • • Cannabis • Signed by President Clinton on October 25, 1994 Previously considered a food, then considered a drug Products intended to supplement health, intended for inges3on, not a conven3onal food, a biologic labeled for health Fell under current Good Manufacturing Prac3ces (cGMP) protocols 5 / 9 The Role of Chromatography Safe Products Raw Material Safe Cul3va3on and Consistent Processing Good Laboratory and Manufacturing Prac>ces (cGxP) Reliable Chromatographic Data • • • • Safety Potency Uniformity Process / Quality Control To make SAFE products, establish GOOD pracFces built on RELIABLE data 6 / 9 Defining Reliable Data Interna3onal Conference on Harmoniza3on (ICH) • • • The ICH has developed guidelines for the valida3on of analy3cal methods (Quality – Q2 (R1)) These approaches can be applied universally Can be used to define “good chromatography” and to guide strong analy3cal methods and prac3ces • • • • • Accuracy Precision Specificity Limit of DetecFon Limit of QuanFtaFon • • • • • Linearity Range Repeatability Intermediate Precision (Ruggedness) System Suitability Range, Linearity and Sensi3vity Response Accuracy Precision LOD = 3:1 S/N Specificity (only molecule in peak) LOQ = 10:1 S/N Concentra3on 8 / 9 Selec3ng Chromatographic Condi3ons Quan,ta,on of Ac,ve ingredients Building a Solid AnalyFcal PlaZorm Potency – Cannabinoids Product Quality and Efficacy Terpenes Quan,ta,on of Residues and Toxins Microbial (E. Coli, Salmonella and Aspergillus) Pes>cide Residues Residual Solvents Heavy Metals 1. Find the technique that fits the data requirements 2. Develop method with simple chromatographic condi3ons 9 / 9 Assessing Technique Fit Ability to provide consistent results and not be affected by common changes Cannabinoids Terpenes PesFcides PesFcides Accuracy Precision LOD / LOQ Linearity Range Specificity Repeatability Robustness System Qualifica>on LC-‐UV √ √ √ √ √ √ √ √ √ GC-‐FID √ √ √ √ √ X √ √ √ LC-‐UV √ √ √ √ √ X √ √ √ GC-‐FID √ √ √ √ √ √ √ √ √ LC-‐MS/ √ √ √ √ √ √ √ √ √ MS/MS GC-‐ √ √ √ √ √ √ √ √ √ LC-‐UV √ √ X √ √ X √ √ √ GC-‐ ECD √ √ √ √ √ X √ √ √ LC-‐UV -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ GC-‐FID √ √ √ √ √ √ √ √ √ MS Solvents 10 / 9 Selec3ng Chromatographic Condi3ons Quan,ta,on of Ac,ve ingredients Building a Solid AnalyFcal PlaZorm Potency – Cannabinoids – LC-‐UV Product Quality and Efficacy Terpenes – GC-‐FID Quan,ta,on of Residues and Toxins Microbial (E. Coli, Salmonella and Aspergillus) Pes>cide Residues – LC-‐MS/MS Residual Solvents – HS-‐GC-‐FID Heavy Metals 1. Find the technique that fits the data requirements 2. Develop method with simple chromatographic condi3ons 11 / 9 The Cannabis Laboratory Today LC -‐UV HS-‐GC-‐FID Quality and Potency • Cannabinoids Quality and Safety • Residual Solvents • Terpenes LC-‐MS/MS Safety • PesFcide Residues 3 techniques to qualify – 3 workflows to opFmize 12 / 9 Potency by LC-‐UV The Workhorse of a QC lab • • • • Least technical instrument to operate High ProducFvity High Sample throughput Accurate quanFtaFon 13 / 9 2014 Emerald Proficiency Test • • • • • • 20 submissions 5 states 4 chromatographic techniques Delta 9 THC in a clean solvent The “true” value for the sample was reported to be 201 ppm. The mean reported value was 212 ppm – Seems to be a bias towards over-‐ repor3ng. – +5.5% GC-‐FID – 221 ppm LC-‐PDA – 211 ppm 14 / 9 Conversion of Acidic Cannabinoids Carboxylic acid Decarboxyla3on GC Inlet Temperatures: 250°C = 482°F Sum of acid and neutral forms of cannabinoids – lack of specificity Conven3onal HPLC HPLC P Speed • • • • ConvenFonal C18, 4.6mm X 150mm X 3.5µm dp Low efficiency Low backpressure Moderate analysis Fme Low equipment cost Wide peaks, Long analyses American Herbal Pharmacopeia, Cannabis Inflorescence, 2013, page 46 Column Construc3on SEM of the Highly Uniform LC Par3cles LC ParFcle with Bonded Phase Comparison of Silica Par3cle Technologies UHPLC 1.9 µm dp 1.9µm dp Fully Porous ParFcle P= Solid, Impermeable Center Φ LηF 2 2 d p dc HPLC 3 µm dp 3 µm dp Lowering the parFcle diffusion by decreasing the parFcle diameter (UHPLC), increases the efficiency of the peak and decreases the run Fme, with the creaFon of backpressure. 18 / 9 Comparison of Silica Par3cle Technologies UHPLC 1.9 µm dp Superficially Porous ParFcle Solid, Solid, Impermeable Impermeable Center Center 1.9µm dp SPP 2.7 µm dp 2.7µm dp Solid, Impermea ble Center Superficially Porous ParFcles can give UHPLC like efficiency and speed with the backpressures of HPLC. 19 / 9 Comparing Efficiency and Pressure of Columns Comparing Pressure and Efficiency of Par3cle Technologies 25000 20000 23411 Raptor SPP Columns exhibit very high efficiency at lower pressures 18682 21211 UHPLC columns exhibit high efficiency with added back pressure Conven3onal FPP par3cles exhibit moderate efficiency 15000 12940 11883 10000 6488 5000 2905 593 775 658 Ultra 3 µm Ultra 5 µm 0 Pinnacle DB 1.9 µm Raptor™ 2.7 µm Raptor™ 5 µm Plates (N) • At optimal linear velocity Pressure (psi) Fast HPLC Analysis of Cannabinoids Speed HPLC Speed (~2700 PSI) P Pes3cides by LC-‐MS/MS The Most Powerful Analy>cal Instrument • • • • • Most technical Most complex Most expensive Most powerful analyFcal tool Extremely sensiFve and versaFle 22 / 9 Mul3ple Reac3on Monitoring -‐ MRM 923 765 532 335 Q1 Q2 Q3 532/335 923/765 Precursor selected Precursor in Q1 Fragmented in Q2 Product Ion Focused in Q3 23 Raptor™ ARC-‐18 -‐ Specifica3ons Stationary Phase Category C18 (L1) Ligand type • Proprietary bonded diisobutyloctadecyl • 7% Carbon Load • Non endcapped Particle • 2.7µm superficially porous • 90Å Pore size • 150m2/g surface area Recommended Usage • pH range 1.0 – 3.0 (usable 1.0-8.0, recommended <2.5) • 80°C Maximum temperature • 600 bar (8,500 psi) operating pressure Recommended Application • Mobile phase is <2.5 pH • High throughput, Multi-component LC-MS analyses • When injection –to -injection RT shifting is observed Pes3cides by LC-‐MS/MS Reproducibility of Problema3c Pes3cides Injection 500 Injection 1 • • 6 Lots of ARC-18 217 Pesticides Abs Diff RT (min) Min = 0.01 Max = 0.07 %Diff 0.29% 3.28% Proof Statement Even after 500 injections, pesticide compounds are well within narrow (15 sec) MRM windows MRM WINDOW Terpenes and Residual Solvents by HS-‐GC Simple and Long-‐las>ng • • • • • Moderately technical Simplified sample preparaFon Inexpensive to operate Very lisle maintenance VersaFle and specialized Terpenes by LC-‐UV :Specificity at 205nm Area of cannabinoid and terpene co-‐elu3on mAU Ch1-205nm,0nm (1.00) 250 MPa 10.0 Cannabinoids Only 9.5 0 9.0 mAU -250 Ch1-205nm,0nm (1.00) 0.5 1.0 MPa 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min Cannabinoid + Terpenes 10.0 250 9.5 0 9.0 mAU Ch1-205nm,0nm (1.00) 0.0 0.5 1.0 MPa 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min Terpenes Only 10.0 250 9.5 0 0.0 9.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min Solu3on versus Full Evapora3on Techniques ParFFoning of VolaFle Analytes G = Gas Phase (headspace) S = Condensed Phase Solvent molecule Solute molecule HEAT (liquid or a solid) Mass Transferred un3l Equilibrium is reached Headspace Analyses: • QualitaFve and QuanFtaFve • Cost EffecFve • Clean Composi3on of a GC Column Polyimide Fused Silica Deac3va3on & Sta3onary Phase mm µm Ligand descrip3on of 624-‐Sil MS 30 / 9 Silarylene Phase Chemistry “5” Phase Back-Bite / Cross-bonded MS Bleed “Sil or MS 5” Phase • Lower bleed for MS • Higher temperature capability Residual Solvents by FET-‐HS-‐GC Selec3vity and reten3on of solvents Terpenes by FET-‐HS-‐GC ~300°C eluFon temperature Summary • Safe products come from sound analyFcal methods and processes • RegulaFon, in some form, is inevitable. Build for that likelihood now by considering industry norms • Choosing the ”best” analyFcal technique can help • Chromatography know-‐how can then be applied to hit the right analyFcal and financial plans Thanks to all the collaborators at Restek 34 / 9
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