RecentImprovements&in&Chromatography:& Advancing ... · Stationary Phase Category C18 (L1) Ligand...
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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
Transcript of RecentImprovements&in&Chromatography:& Advancing ... · Stationary Phase Category C18 (L1) Ligand...
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
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What will happen to Cannabis tes3ng?
FOOD?
DRUG?
LEGAL?
ILLEGAL?
WHO AM I?
SUPPLEMENT? INGREDIENT?
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Regula3ons
Code of CO RegulaFons
Retail
Marijuana Code
Colorado Marijuana
Enforcement Division (MED)
State RegulaFons
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
Federal LegalizaFon Federal Oversight
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Has a precedent been set?
Dietary Supplements Health and Educa3on Act of 1994 (DSHEA)
• 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
Ginseng
Cannabis
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The Role of Chromatography
Safe Products Raw Material
Safe Cul3va3on and Consistent Processing
Reliable Chromatographic Data
Good Laboratory and Manufacturing Prac>ces (cGxP) • Safety
• Potency • Uniformity • Process / Quality
Control
To make SAFE products, establish GOOD pracFces built on 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
Defining Reliable Data
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Range, Linearity and Sensi3vity Response
Concentra3on
LOD = 3:1 S/N
LOQ = 10:1 S/N
Precision Accuracy
Specificity (only molecule in peak)
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Selec3ng Chromatographic Condi3ons
Quan,ta,on of Ac,ve ingredients 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
Building a Solid AnalyFcal PlaZorm
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Accuracy Precision LOD / LOQ Linearity Range Specificity Repeatability Robustness System Qualifica>on
√ √ √ √ √ √ √ √ √
√ √ √ √ √ X √ √ √
√ √ √ √ √ X √ √ √
√ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √
√ √ X √ √ X √ √ √
√ √ √ √ √ X √ √ √
-‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐
√ √ √ √ √ √ √ √ √
Cannabinoids LC-‐UV
GC-‐FID
Terpenes LC-‐UV
GC-‐FID
PesFcides
LC-‐MS/MS
GC-‐MS/MS
Solvents LC-‐UV
GC-‐FID
PesFcides LC-‐UV
GC-‐ECD
Assessing Technique Fit Ability to provide consistent results and not be affected by common changes
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Selec3ng Chromatographic Condi3ons
Quan,ta,on of Ac,ve ingredients 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
Building a Solid AnalyFcal PlaZorm
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The Cannabis Laboratory Today
Quality and Potency • Cannabinoids
LC -‐UV HS-‐GC-‐FID LC-‐MS/MS
Quality and Safety • Residual Solvents • Terpenes
Safety • PesFcide Residues
3 techniques to qualify – 3 workflows to opFmize
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Potency by LC-‐UV
The Workhorse of a QC lab • Least technical instrument to operate • High ProducFvity • High Sample throughput • Accurate quanFtaFon
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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
Conversion of Acidic Cannabinoids
GC Inlet Temperatures: 250°C = 482°F
Carboxylic acid
Decarboxyla3on
Sum of acid and neutral forms of cannabinoids – lack of specificity
Conven3onal HPLC
HPLC
• Low efficiency • Low backpressure • Moderate analysis Fme • Low equipment cost
P Speed
American Herbal Pharmacopeia, Cannabis Inflorescence, 2013, page 46
ConvenFonal C18, 4.6mm X 150mm X 3.5µm dp
Wide peaks, Long analyses
Column Construc3on
SEM of the Highly Uniform LC Par3cles
LC ParFcle with Bonded Phase
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Comparison of Silica Par3cle Technologies
1.9µm dp
3 µm dp
Fully Porous ParFcle
Solid, Impermeable
Center
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.
1.9 µm dp
3 µm dp
UHPLC
HPLC
22cp ddFLP ηΦ
=
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Comparison of Silica Par3cle Technologies
1.9µm dp Superficially Porous ParFcle
Solid, Impermeable
Center
Superficially Porous ParFcles can give UHPLC like efficiency and speed with the backpressures of HPLC.
1.9 µm dp
2.7 µm dp
UHPLC
SPP Solid,
Impermeable Center
Solid, Impermea
ble Center
2.7µm dp
Comparing Efficiency and Pressure of Columns
21211
23411
18682
11883 12940
6488
2905
593 775 658
0
5000
10000
15000
20000
25000
Pinnacle DB 1.9 µm Raptor™ 2.7 µm Raptor™ 5 µm Ultra 3 µm Ultra 5 µm
Comparing Pressure and Efficiency of Par3cle Technologies
Plates (N) Pressure (psi)
• At optimal linear velocity
UHPLC columns exhibit high efficiency with added back pressure
Conven3onal FPP par3cles exhibit moderate efficiency
Raptor SPP Columns exhibit very high efficiency at lower pressures
Fast HPLC Analysis of Cannabinoids Speed
(~2700 PSI)
HPLC
P Speed
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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
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Q1 Q2 Q3
Precursor selected in Q1
Precursor Fragmented in Q2
Product Ion Focused in Q3
923/765
765
335
923
532
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Mul3ple Reac3on Monitoring -‐ MRM
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 1
MRM WINDOW
Injection 500
• 6 Lots of ARC-18 • 217 Pesticides
Abs Diff %DiffRT (min)
Min = 0.01 0.29%Max = 0.07 3.28%
Proof Statement Even after 500 injections, pesticide compounds are
well within narrow (15 sec) MRM windows
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
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
0
250
mAU
9.0
9.5
10.0
MPaCh1-205nm,0nm (1.00)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
0
250
mAU
9.0
9.5
10.0
MPaCh1-205nm,0nm (1.00)
Cannabinoids Only
Cannabinoid + Terpenes
Terpenes Only
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min-250
0
250
mAU
9.0
9.5
10.0
MPaCh1-205nm,0nm (1.00)
Area of cannabinoid and terpene co-‐elu3on
Solu3on versus Full Evapora3on Techniques
ParFFoning of VolaFle Analytes
G = Gas Phase (headspace)
S = Condensed Phase (liquid or a solid)
Mass Transferred un3l Equilibrium
is reached
Solute molecule
Solvent molecule
HEAT
Headspace Analyses: • QualitaFve and
QuanFtaFve • Cost EffecFve • Clean
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Composi3on of a GC Column
mm
µm
Polyimide Fused Silica
Deac3va3on & Sta3onary Phase
Ligand descrip3on of 624-‐Sil MS
“5” Phase
“Sil or MS 5” Phase Back-Bite / Cross-bonded
MS Bleed
Silarylene Phase Chemistry
• 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
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Summary
Thanks to all the collaborators at Restek
• 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
