GC  Methods  for  Cannabis  Safety  and  Potency  Tes6ng  

Amanda  Rigdon1  ,  Jack  Cochran1  ,  Corby  Hilliard1  ,  William  Schroeder2  ,  Chris<  

Schroeder2  ,  Theo  Flood2    

1  Restek,  Bellefonte,  PA,  USA,  2Cal-­‐Green  Solu<ons,  San  Luis  Obispo,  CA,  USA  

Outline

•  Residual  solvent  analysis  –  Mi<ga<ng  matrix  effects  –  Confirma<on  column  

•  Pes<cide  analysis  –  QuEChERS  sample  prep  –  Recovery  studies  –  Detec<on  methods  

•  Potency  analysis  –  GC  vs.  LC  repor<ng  –  Quan<ta<ve  Bias  

How  do  we  measure  residual  solvents?

 • Confirma<on  and  quan<fica<on  of  residual  solvents  in  pharmaceu<cals  

•   GC-­‐HS-­‐FID  methodology  

•   Depends  on  accurate  repor<ng  of  solvents  used  during  manufacture  

Residual  Solvents  in  Cannabis  Concentrates

Butanes  Heptanes  Benzene  Toluene  Hexane  Xylene  Ethanol  

Isopropanol  Acetone  

                           Par<<oning  of  Vola<le  Analytes  

G  =  Gas  Phase        (headspace)  

S  =  Condensed  Phase    

(liquid  or  a  solid)  

   HEA

T   Mass  Transferred    un6l  Equilibrium    is  reached  

Solute molecule

Solvent molecule

Introduc:on  to  HS-­‐GC

Matrix  Effects

Equilibriu

m  Quan<fica<on  in  HS-­‐GC  depends  upon  the  establishment  of  equilibrium  in  a  par<<oning  system.  Difficult  matrices  can  introduce  adsorp<on  effects  or  change  par<<on  coefficients  of  

analytes  of  interest.    

Introduc:on  to  FET-­‐HS-­‐GC

In  this  example,  a  saltwater  matrix  will  dras<cally  decrease  the  par<<on  

coefficients  of  some  solvents,  infla<ng  results  unless  matrix-­‐matched  

standards  are  used.  

Matrix  Effects

The  complex  nature  of  cannabis  concentrates  is  likely  to  give  rise  to  matrix  effects,  which  may  reduce  quan<ta<ve  accuracy.  Addi<onally,  given  the  variety  of  concentrates  

available,  solubility  in  solvents  that  do  not  interfere  with  later  elu<ng  

analytes  of  interest  (e.g.  xylenes)  may  be  an  issue.  

?  

Mi:ga:ng  Matrix  Effects  –  USP  <467>

Procedure  A:  Screening  Dissolved  sample  analyte  peak  areas  are  compared  to  standard  

peak  areas  at  cutoff  

Procedure  B:  Confirma<on  Dissolved  sample  analyte  peak  areas  are  compared  to  standard  peak  areas  at  cutoff  on  a  column  

with  alternate  selec<vity  

Procedure  C:  Quan<fica<on  Dissolved  sample  analyte  peak  areas  are  compared  to  matrix-­‐matched  standard  peak  areas  at  cutoff  

Quan<fica<on  is  corrected  for  matrix  interferences  by  using  matrix  matched  standard.  

Mi:ga:ng  Matrix  Effects  The  full  evapora:on  technique  (FET)

Mi:ga:ng  Matrix  Effects  -­‐  FET

Increase  par<<oning  efficiency  by  increasing  surface  area  of  the  solid  sample  –  140°C  for  30  minutes.  

Photos  and  mel3ng  point  data  courtesy  of  Cal-­‐Green  Solu3ons  

Confirma:on  Columns

3  

2  

1  4  

5  

67  

624-­‐type  (G43)    column  

1)  Methanol  2)  Pentane  3)  Ethanol  4)  Hexane  5)  Benzene  

6)  Heptane  7)  Toluene  8/9)  m,p-­‐xylene  10)  o-­‐xylene  

8/9  

10  

Wax-­‐type  (G16)  column  

4)  Hexane  2)  Pentane  6)  Heptane  1)  Methanol  5)  Benzene  

3)  Ethanol  7)  Toluene  8)  p-­‐xylene  9)  m-­‐xylene  10)  o-­‐xylene  

42

6 15

3

7

89

10  

Chromatograms from EZGC chromatogram creator www.restek.com/ezgc

Pes:cide  Analysis

The  QuEChERS  Process

Sample  Homogeniza<on  and  Weing  

Extrac<on  

Cleanup  

Reduce  sample  par<cle  size  to  improve  extrac<on  efficiency.  Use  a  homogenizer  or  cryo-­‐mill.  Wet  sample  so  it  contains  >  80%  water  

QuEChERS  extrac<on  is  a  liquid-­‐liquid  extrac<on  that  effects  par<<oning  using  salts.  This  step  should  extract  most  analytes,  as  well  as  many  matrix  components  (e.g.  chlorophyll)  

Matrix  components  are  removed  from  the  extract  using  either  dispersive  sorbents  or  cleanup  cartridges.    

The  QuEChERS  Process  Extrac:on

Extract  

Solid  Matrix  

Water  +  Matrix  

Salt  

The  QuEChERS  Process  Cleanup

Performing  a  Preliminary  Recovery  Study

•  Prepare spiked samples at a mid-level concentration by adding a known amount of analyte to the sample prior to extraction.

•  Extract spiked samples •  Extract blank samples •  Spike final extract from blank samples with analyte constituting

100% recovery •  Analyze the pre and post-extraction spiked samples.

%  Recovery  =    Analyte  area  in  pre-­‐extrac<on  spike  

Analyte  area  in  post-­‐extrac<on  spike  

X  100  

Recovery  Study  Requirements

•  Use internal standards for quantificaton

•  Use matrix-matched standards for each commodity

•  Evaluate recoveries at low, mid, and high levels

•  Multiple replicates at each level required

•  Make sure to also analyze unspiked blank matrix for interferences!

Pes:cide  Analysis

THC?  

CBD?  

LC-­‐MS/MS  may  suffer  from  severe  ion  

suppression  due  to  co-­‐elu<ng  

cannabinoids  

While  GC-­‐MS/MS  is  less  prone  to  ion  suppression,  

interference  may  s<ll  occur.  

204  pes<cides  in  7  minutes!  

Why  Test  Potency  by  GC?

•  GCs  are  less  expensive  to  purchase,  maintain,  and  operate  

•  Standards  as  less  expensive  •  Separa<on  is  more  

straighporward  (fewer  compounds)  

PROCESS  MONITORING!  

High  CBD?  High  THC?  

Mismatch  between  GC  and  LC  Potency  Results

LC  Results:  18%  THCA,  1%  THC  

GC  Results:  

13.3%  loss  of  mass  in  the  GC  inlet  for  THCA  

due  to  loss  of  carboxylic  acid  group.    

16.8%  THC:  (18  *  0.877)  +  1  

Calcula:ng  Decarboxyla:on  Efficiency

min 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Inject  equal  concentra3ons  THC  and  THCA  standards  (solvent  or  spiked  matrix  standards)  

THC  Standard  area  =  40.2  pA*s  

THCA  Standard  area  =  22.8  pA*s  

THCA  area  Percent  (%ATHCA)  =  (22.8/40.2)*100  =  56.7%  

At  100%  decarboxyla<on  efficiency,  %ATHCA  should  be  87.7%.  

Decarboxyla<on  efficiency  =  (ATHCA  /  87.7)  *100  =  64.7%  

Op:mizing  GC  Inlet  for  Potency  Analyses

min 2.3 2.35 2.4 2.45 2.5 2.55 2.6 2.65 2.7

100%  decarb.  efficiency  

Base  deact.  liner,  double  wool.  280°C,  20:1  split  84.0%  efficiency  

IP  deact.  liner  w/wool.  280°C,  20:1  split  

64.6%  efficiency  

Op:mizing  GC  Inlet  for  Potency  Analyses

min 2.2 2.3 2.4 2.5 2.6 2.7

100%  decarb.  efficiency  Base  deact.  liner,  double  wool.  300°C,  10:1  split  

Base  deact.  liner,  double  wool.  320°C,  10:1  split  

91.9%  efficiency  

90.7%  efficiency  

So  Everything’s  Great,  Right? Decarboxyla<on  efficiency  seems  to  change  over  <me  and  between  commodi<es.  Need  to  determine  if  the  method  

performs  consistently.  

Ini<al  decarb.  efficiency:  82.3%  Ater  6  calibra<on  curves:  73.6%  

Ater  100  standard  injec<ons:  45.8%  Ater  200  standard  injec<ons:  30.0%  

Decarboxyla<on  efficiency  over  <me  with  base  deac<vated  liner  

Summary

•  Either  USP  <467>  or  the  full  evapora<on  technique  are  suitable  for  analysis  of  residual  solvents  in  cannabis  concentrates  –  Matrix  matched  standards  should  be  used  –  Confirma<on  should  be  performed  on  a  column  with  alternate  selec<vity  

•  QuEChERS  is  a  proven,  fast,  and  simple  method  of  sample  prepara<on  for  pes<cide  analyses  –  Co-­‐elu<ng  cannabinoids  will  be  problema<c  –  LC-­‐MS/MS  may  suffer  from  ion  suppression  

•  Decarboxyla<on  efficiency  for  potency  analyses  must  be  considered.    –  Increase  efficiency  by  increasing  inlet  temperature  and  analyte  residence  

<me  

Ques6ons?