Transcript slide template - National Forensic Science Technology

Technology Transition Workshop
DART Workshop – Ames, IA
August 2008
Use of the AccuTOF-DART system for
the forensic analysis of drugs of abuse
Bob Steiner
Virginia Department of Forensic Science
Central Laboratory
Richmond, VA
804-786-4707 x22347
[email protected]
Technology Transition Workshop
GLOSSARY OF TERMS:
 AccuTOF – Accurate mass Time Of Flight; mass spectrometer
 DART – Direct Analysis in Real Time; atmospheric pressure ion source
 Orifice 1 – inlet to the AccuTOF; raising voltage causes fragmentation
of ions via collision induced dissociation
 Profile Spectrum – multiple data points to describe the mass peaks of
a spectrum; have “chromatogram” appearance
 Centroid – to find the center of a mass peak collected in profile mode;
gives resulting histogram spectrum as a mass vs. abundance pair
 PEG600 – polyethylene glycol polymer with an average MW of 600
Da; used for internal mass calibration
 Internal mass calibration – PEG600 spectrum run within a data file;
allows to accurately set the mass axis for all spectra in that file
Technology Transition Workshop
GLOSSARY OF TERMS:
 Function switching – method that allows changing the orifice 1
voltage rapidly during data collection; ori 1 voltage changes every 0.25
sec
 Protonated or deprontonated molecule – the addition or subtraction
of a hydrogen from the neutral molecule, depending on polarity of DART
 Millimass unit (mmu) – accuracy of TOF spectra measured to
thousandths of a Dalton; acceptable spectra are < 5 mmu from calculated
mass
 JEOL-DX file – text file of data points in a mass spectrum; can be
easily exported to other software for analysis
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GLOSSARY OF TERMS:
 SearchFromList – library searching program; searches by
empirical formula and matching user-library spectra
 Drugs_Neutral_Masses – library of empirical formulae of drugs
 Drug Prep Library_ori20 – library spectra of pharmaceuticals at orifice 1
voltage of 20 V; also libraries at ori30, 60 and 90 V
 Drug Std Library_ori20 – library spectra of primary standards at orifice 1
voltage of 20 V; also libraries at ori30, 60 and 90 V
 Mirror spectrum – display of unknown (positive) and known (negative)
spectra in SearchFromList program
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DART METHODOLOGY
How do I use the AccuTOF-DART for
drug samples?
 Types of samples
 Best sampling methods
 Function Switching
 Calibration considerations
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HEADSPACE SAMPLING
1. Start acquisition
2. Open container near DART gas stream
3. Remove container
Can’t get simpler than that!!!!!!
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SAMPLING OF SOLIDS
Hold solid material in DART gas stream using tweezers
Drawbacks:
1. Hard to hold
2. MESSY
3. Non-homogeneous
samples
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MORE SOLID SAMPLING DRAWBACKS
Coated
tablets!
GAS STREAM IS HOT!!!
4-chloro-2,5-dimethoxyamphetamine
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“DRIED ON TUBE” METHOD
Deposit sample onto tubes
with syringe
Can measure how much
sample is deposited
Turned out not so good!
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“DRIED ON TUBE” METHOD
Loading cap tubes with sample
Even 1 uL “runs down” tube
Inconsistent results obtained
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Injecting sample into gas
stream with syringe
Draw up samples from ALS vials
Can measure amount injected
Turned out not so good!
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LIQUID SAMPLING
Dissolve sample into suitable solvent
Dip capillary tube in liquid/hold “wand” in DART gas stream
Advantages:
1. EASY!!!
2. Can control concentration
of sample
3. Homogeneous sample
Preferred method at DFS!!!
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DART sampling
by hand is an
ART!
Consistency from one
person to the next is
difficult to obtain!!!
Buy an AutoDART???
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FUNCTION SWITCHING
In general, ionization of molecules with DART
produces hydride addition or subtraction products
of the molecular ion.
Methyl stearate spectrum showing [M+H]+ ion at 299.2931 Da.
No ‘diagnostic’ ions
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Collision-Induced Dissociation
 Definition*: “An ion/neutral species interaction wherein the
projectile ion is dissociated as a result of interaction with a target
species. This is brought about by conversion of part of the
translation energy of the ion to internal energy in the ion during
collision.”
 High vacuum prevents this in EI
 Basis behind triple stage quadrupole systems
 Commonly done in LCMS systems to promote fragmentation
 Performed in the AccuTOF by raising orifice 1 voltage
*http://mass-spec.lsu.edu/msterms/index.php/Collision-Induced_Dissociation
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EFFECT OF RAISING ORIFICE 1 VOLTAGE
Orifice 1 = 15V
Orifice 1 = 30V
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EFFECT OF RAISING ORIFICE 1 VOLTAGE
Orifice 1 = 45V
Orifice 1 = 60V
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Function
Switching
allows
collection
of data at
SEVERAL
orifice 1
voltages
AT ONCE!
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Noscapine
Orifice 1 = 20
Orifice 1 = 30
Orifice 1 = 60
Orifice 1 = 90
Function
Switching
results
showing
fragmentation
at various
orifice 1
voltages
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RUNNING A SAMPLE ON THE AccuTOF-DART
TIC[2]; / ESI+ / cal7908
3
x10
Lock and
chk stds
Intensity (715231)
PEG
0.258
1.007
More PEG
1.157
2.554
Sample
0.674
500
2.221 2.438
1.839
0.425
1.656
1.972
0
0
0.5
1.0
1.5
Time[min]
2.0
2.5
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Mass calibrations
Calibration at acquisition –
PEG+H
Internal mass calibration –
temp_PEG
Set at tune (after cleaning)
Within your data file
“Global” mass calibration
“Fine tunes” your calibration
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“Run PEG before
AND/OR after samples
within your datafile!
Intensity matters!!”
Peggy the Dimetrodon
TIC[2]; / ESI+ / jhp_310a_311a
3
x10
PEG
Intensity (600571)
PEG
2.486
3.085
500
0.705
0.971
0.389
1.920
1.3871.637
0
0
1.0
2.0
Time[min]
3.0
3.317
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GOOD PEG vs BAD PEG
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TIC[2]; / ESI+ / cal1308
3
x10
Intensity (809700)
Where to
average your
peak for best
PEG intensity?
TIC
0.474
500
0.291
0.674
0.524 0.558
0
0.30
0.40
0.50
Time[min]
0.60
0.70
MS[2];0.442..0.493;-1.0*MS[2];0.361..0.415; / ESI+ / cal1308
profile
Intensity (6120)
371.22813
5000
30V
data
459.27946
503.30537
327.19959
195.12221
239.14916 283.17463
547.33420
89.05961 133.08629
0
100
200
300
400
500
415.25460
459.27969
600
m/z
MS[2];0.442..0.493;-1.0*MS[2];0.361..0.415; / ESI+ / cal1308
3
x10
centroid
Area (23621)
20
503.30555
327.19988
10
195.12257
239.14967 283.17499
547.33415
87.04568 133.08689
0
100
200
300
400
m/z
500
600
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TIC[2]; / ESI+ / cal1308
3
x10
Intensity (809700)
TIC
0.474
Less intensity!
500
0.291
0.674
0.524 0.558
0
0.30
0.40
0.50
Time[min]
0.60
0.70
MS[2];0.429..0.571;-1.0*MS[2];0.356..0.412; / ESI+ / cal1308
profile
Intensity (3373)
415.25622
503.30599
547.33324
327.20019
2000
195.12242
239.14952 283.17523
89.05965 133.08647
0
100
200
300
400
500
600
m/z
MS[2];0.429..0.571;-1.0*MS[2];0.356..0.412; / ESI+ / cal1308
3
x10
centroid
Area (14357)
415.25632
503.30621
547.33319
10
327.20052
195.12281
239.15015 283.17553
87.04657 133.08721
0
100
200
300
400
m/z
500
600
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Centroiding peaks = difficult with low intensity ions
MS[2];3.532..3.699;-1.0*MS[2];3.367..3.512; / ESI+ / jhp_272a_274a
Intensity (659)
133.09083
500
Interfering
peak or noise
0
133.00
133.10
m/z
133.20
MS[2];3.532..3.699;-1.0*MS[2];3.367..3.512; / ESI+ / jhp_272a_274a
Intensity (3161)
“tailing” due to kinetic
energy spread in TOF
415.25405
2000
0
415.20
415.40
m/z
415.60
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LOCK MASS AND CHECK STANDARDS
 Mix of cocaine, methamphetamine and nefazodone
 MWs span the mass range
 Cocaine is used as drift compensation “lock” at 304.1549 Da
 Methamphetamine (150.1283 Da) and Nefazodone
(470.2323 Da) cover the low and high ends of mass range
 Adds another level of calibration applied to sample data
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LOCK MASS AND CHECK STANDARDS
Cocaine 304.1549
304.1549
100
Rel. Abundance
80
60
40
Meth 150.1283
150.1296
Nefazodone 470.2323
20
470.2362
0
120
160
200
240
280
m/z
320
360
400
440
480
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SAMPLE DATA
TIC[2]; / ESI+ / af070701
3
x10
Sample signal is
averaged,
centroided and
calibrations are
applied for EACH
orifice voltage
Intensity (296800)
200
2.270
2.919
2.537
2.853
0
2.0
2.5
Time[min]
3.0
Spectra are saved in “JEOL-DX” format
MS[2];2.225..2.317;-1.0*MS[2];2.042..2.192; / ESI+ / af070701
3
x10
Area (15434)
324.20773
192.08031
10
325.21241
192.17013
87.10397
192.24602
0
100
150
223.09892
200
250
m/z
324.48445
340.22848
300
350
400
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SearchFromList Program – by Dr. Robert “Chip” Cody
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Match spectra search
Load data file
Load empirical
formula library
Adjust these to
suit your search
Empirical formula
search
Add or subtract from empirical formula
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SearchFromList empirical
formula search result
Spectrum file name
From empirical
formula library
Lot number of std
used to create lib
spectrum
must be < 5 mmu
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SearchFromList empirical
formula search result
Search result label
Spectrum imported from Mass Center
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Match spectra search
Choose library to match orifice 1
voltage for the spectrum you are
searching
DOUBLE CLICK on library
entry to load all spectra!
Search!
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Match spectra search results
Blue = original spectrum
Click on entries to view
comparison spectra
Red = comparison spectrum
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Suggested Data for casefiles
• Print out: TIC[2] from Chromatogram view
• Print out spectrum of cocaine lock mass and check
stds
• Save spectra as averaged, background subtracted,
centroided, calibrated JEOL-DX files
• Print out: SearchFromList search result for 20V
spectrum
• Print out: Other spectra or search results at other
voltages – enough to characterize the spectrum
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FUNCTION SWITCHING - ADVANTAGES
 FOUR spectra collected every second
 Higher orifice 1 voltages result in more fragmentation
 Can lead to greater confidence for identification
 Except for mixtures, spectra are reproducible
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FUNCTION SWITCHING - DISADVANTAGES
 Mixture spectra – no prior chromatography (can be good
OR bad!!)
 Less sensitivity – splitting ionization between four functions
 Finite database for searching – library databases need to
be developed!
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Application of the DART to DX
 Currently being used as a
screening tool for DX casework.
 Validation took over one year to
complete.
 Approved by DFS Scientific
Advisory Board and full VA
Forensic Science Board, May 6-8,
2008.
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AccuTOF-DART Validation at DFS
1. DART screening method - LLOD determined for 7 drugs; daily
calibration data to show stability of instrument
2. DART sampling methods – why we use methanol and MP tube
‘wands’
3. Comparison of DART to GCMS – new technology vs.
established technology; 553 samples run on each – ALL match in
highest Scheduled drug found; DART typically showed more
cmpds!
4. Selectivity study – can you tell the difference in DART spectra of
drugs with the SAME empirical formula??
5. “Rugustness” – same result on different days with different
people??
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LLOD DETERMINATION
and INSTRUMENT STABILITY
Conc.
Drug Name
0.5 mg/mL
7/13/2007
0.1 mg/mL
7/13/2007
Exact Mass
measured mass
difference
Alprazolam
309.0907
309.0906
0.0001
Butalbital
225.1239
225.1240
0.0001
Cocaine
304.1549
304.1526
0.0023
Heroin
370.1654
370.1636
0.0018
Methamphetamine
150.1283
150.1268
0.0033
Testosterone Propionate
345.2430
345.2409
0.0021
Trazodone
372.1591
372.1553
0.0038
Alprazolam
309.0907
309.0888
0.0019
Butalbital
225.1239
225.1246
0.0007
Cocaine
304.1549
304.1545
0.0004
Heroin
370.1654
370.1670
0.0016
Methamphetamine
150.1283
150.1295
0.0012
Testosterone Propionate
345.2430
345.2427
0.0003
Trazodone
372.1591
372.1626
0.0035
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COMPARISON OF DART RESULTS WITH
ANALYTICAL SCHEME – 553 SAMPLES!
DART result
GCMS confirmation
1161 heroin, papaverine,
6-MAM, quetiapine
1161 heroin
1665 cocaine, diltiazem,
naproxen
1665 cocaine
1910 MDMA, caffeine,
procaine
1910 MDMA, caffeine, procaine,
dimethylsulfone
1976 cocaine, caffeine,
benzocaine
1976 cocaine, caffeine
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SELECTIVITY STUDY
119.0872
Ori1 30V
Easily distinguish methamphetamine from phentermine!
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SELECTIVITY STUDY
Cocaine and scopolamine
ori1 30V
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SELECTIVITY STUDY
Cocaine and scopolamine
ori1 90V
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SELECTIVITY STUDY
LSD and LAMPA
ori1 90V
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GHB SCREENING
“Detection of GHB in various drink matrices via AccuTOF-DART”
Bennett MJ, Steiner RR. J. Forensic Sci., in press*
Spiked 50 beverages with GHB at
“impairment levels” to determine if
DART could detect
GHB easily seen in all drink matrices
Gave better results than using GHB
color test!
Done in NEGATIVE ion mode
* slated for publication Jan 2009
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GHB SCREENING
blank
[GHB-H]-
Ocean Spray Cranberry Juice
- blank
Ocean Spray Cranberry Juice
– 2 mg/mL spike with GHB
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DART Spectral
Interpretation
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Molecular Weight Information
Electron
Impact
Ionization
DART
Ionization
• M+.
 [M+H]+ or [M-H]-
•Unstable – leads to extensive
fragmentation
Very stable
•Usually the peak @ highest
mass, excluding isotope peaks
•Look for “logical neutral
losses”
• Sometimes hard to find!
Very little fragmentation
(without help!)
Proper terms: Protonated
or deprotonated molecule
Loss of stable neutrals
 Use SearchFromList to find
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Abundance
#
3
7
2
:
V
E
R
A
P
A
M
3
I L - - D
0 3
9500
9000
I P
DART Spectral
Characteristics
8500
8000
7500
EI - DIP
7000
6500
6000
5500
5000
verapamil – MW 454.61 Da
4500
4000
3500
3000
2500
2000
1500
5
1
8
5
1
1000
2
500
8
41
0
7
1
3
0
1
7
7
2
1
6
0
8
4
0
40
60
5
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z-->
DART
3
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DART Spectral
Characteristics
Oxytetracycline
MW 460.44 Da
Stable
neutral loss
of H2O
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All the usual
isotope clusters!!
DART Spectral
Characteristics
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Methamphetamine
DART Spectral
Characteristics
CH2
CH3
CH
H
+
N
H
CH3
- NH2CH3
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DART Spectral
Characteristics
CH3
Phentermine
CH2
CH3
C
+
NH2
H
- NH3
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DART Spectral
Characteristics
CH3
Heroin
H
N
+
CH3
CH2
CH
_
CH2
OH
+ CH2
C
C
O
O
CH2
CH3
CH3
C
O
O
_
CH3
O
O
OH
C
C
O
O
NOT!
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DIMERS and
Toto, too!!
TRIMERS and
ADDUCTS!!!
OH MY!!!!!!!!!
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Dimers – hydrocodone/APAP (Lortab)
Ori1 = 20V
[M+H]+
Ori1 = 20V
dimer
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C
O
CH
3
Acetaminophen dimer
NH
+
O
H
H
19.1%
O
OH
C
NH
CH3
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Dimers – hydrocodone/APAP (Lortab)
Ori1 = 30V
Ori1 = 60V
Dimer
disappears!
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Elemental Composition Calculation
1/3/2008
10:08:11 AM
Element Limits:
C 0/40 H 0/50 N 0/10 O 0/10
Tolerance: 5.00 mmu
Even or odd electron ion or both: BOTH
Minimum unsaturation: -0.5
Maximum unsaturation: 100.0
Meas. mass
u
451.227203
C18H21NO3
+ C8 H9 N O2
Abund. Diff.
%
mmu
0.00
3.91
Hyd or Cod
APAP
[C26H30N2O5 + H] adduct!!
Unsat.
Compositions
12.5
C26 H31 N2 O5
WHAT’S THIS?
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Salsalate (disalicylic acid) – analgesic,
anti-inflammatory
OH
121
O
MW = 258.0528 Da
C
O
C
O
OH
137
In MEOH, ori1=20V
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Salsalate – ammonium adduct
NH4OH on swab in DART sampling
area
[M+NH4–H2O]+ = 258.0766 Da
[M+H]+
MW of Salsalate = 258.0528 Da
Emp Form: C14H10O5
[salicylic acid+NH4–H2O]+
Charge carrier
+NH4 in SFL
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Salsalate – ammonium adduct
276.086517
16.63
-2.52
2.01
2.00
0.66
-0.67
-0.67
1.0
9.5
4.0
9.0
14.0
8.5
C1 H12 N10 O7
C10 H10 N7 O3
C11 H16 N0 O8
C12 H12 N4 O4
C13 H8 N8 O0
C14 H14 N1 O5
[C14H10O5 + NH4]+
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Salsalate – ammonium adduct
Elemental composition
Element Limits:
C 0/40 H 0/50 N 0/10 O 0/10
Tolerance: 5.00 mmu
Even or odd electron ion or both: BOTH
Minimum unsaturation: -0.5
Maximum unsaturation: 100.0
Meas. mass
u
138.053833
Abund.
%
15.72
Diff.
mmu
1.01
-0.33
-1.67
Unsat.
Compositions
0.0
5.0
4.5
C4 H10 N0 O5
C5 H6 N4 O1
C7 H8 N1 O2 [C7H6O3 + NH4 – H2O]+
258.072021
15.47
-1.93
-3.27
-4.61
3.94
5.0
10.0
9.5
14.0
C11 H14 N0 O7
C12 H10 N4 O3 NOT M+. but
C14 H12 N1 O4
C18 H10 N0 O2 [C14H10O5+NH4–H2O]+
259.063446
12.03
2.81
2.80
1.45
0.13
15.0
9.5
14.5
14.0
C13 H5 N7 O0
C14 H11 N0 O5
C15 H7 N4 O1
C17 H9 N1 O2
[C14H10O5+H]+
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Salsalate – ammonium adduct
OH
[3(C7H4O2)+ NH4]+
O
C
O
C
O
“Salicylate” dimer
[2(C7H4O2) + H]+
“Salicylate”
[C7H4O2 + H]+
OH
“Salicylate” trimer
[3(C7H4O2) + H]+
[3(C7H4O2) + H2O + NH4]+
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Salsalate – ammonium adduct
Elemental composition
Element Limits:
C 0/40 H 0/50 N 0/10 O 0/10
Tolerance: 5.00 mmu
Even or odd electron ion or both: BOTH
Minimum unsaturation: -0.5
Maximum unsaturation: 100.0
Meas. mass
u
361.068268
Abund.
%
7.36
Diff.
mmu
-2.94
-2.94
2.92
Unsat.
Compositions
21.0
15.5
24.5
C20 H7 N7 O1
C21 H13 N0 O6
C28 H9 N0 O1
378.095795
15.06
-0.61
-1.96
-1.99
-4.64
3.89
15.0
20.0
14.5
19.0
23.5
C19 H14 N4 O5
C20 H10 N8 O1
C21 H16 N1 O6 [3(C7H4O2 + NH4]+
C24 H14 N2 O3
C28 H12 N1 O1
396.108185
7.08
-0.14
-0.14
-1.50
-2.82
-4.15
19.0
13.5
18.5
18.0
23.0
C20 H12 N8 O2
C21 H18 N1 O7 [3(C7H4O2)+H2O+NH4]+
C22 H14 N5 O3
C24 H16 N2 O4
C25 H12 N6 O0
“Salicylate” trimer
[3(C7H4O2) + H]+
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CH2
Cl
N
N
CH2
O
CH2
CH2
OH
What is this???
CH
Hydroxyzine (Vistaril)
[M+H]+: 375.1839 Da
In MEOH, ori1 20V
Chlorinated!
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Elemental Composition Calculation
12/12/2007 11:25:13 AM
Element Limits:
C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1
Tolerance: 5.00 mmu
Even or odd electron ion or both: BOTH
Minimum unsaturation: -0.5
Maximum unsaturation: 100.0
Meas. mass
u
537.233582
Abund. Diff.
%
mmu
5.76
3.53
2.19
0.84
-0.50
-0.50
-1.84
-3.15
-3.19
-4.50
4.05
2.70
-1.32
Elemental composition
NOW WHAT?????
Unsat.
Compositions
6.5
6.0
5.5
10.5
5.0
10.0
15.0
9.5
14.5
19.0
18.5
22.5
C18 H34 N10 O7 Cl1
C20 H36 N7 O8 Cl1
C22 H38 N4 O9 Cl1
C23 H34 N8 O5 Cl1
C24 H40 N1 O10 Cl1
C25 H36 N5 O6 Cl1
C26 H32 N9 O2 Cl1
C27 H38 N2 O7 Cl1
C28 H34 N6 O3 Cl1
C32 H32 N5 O1 Cl1
C34 H34 N2 O2 Cl1
C39 H34 N0 O0 Cl1
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CH2
Cl
N
N
CH2
O
CH2
CH2
OH
CH
Hydroxyzine empirical formula
C21H27N2O2Cl
Look at composition list
and see if any are
“terribly unreasonable”
Technology Transition Workshop
Elemental Composition Calculation
12/12/2007 11:25:13 AM
Element Limits:
C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1
Tolerance: 5.00 mmu
Even or odd electron ion or both: BOTH
Minimum unsaturation: -0.5
Maximum unsaturation: 100.0
Meas. mass
u
537.233582
Hydroxyzine empirical formula
C21H27N2O2Cl
Apply Chemistry logic!!
Abund. Diff.
%
mmu
5.76
3.53
2.19
0.84
-0.50
-0.50
-1.84
-3.15
-3.19
-4.50
4.05
2.70
-1.32
Unsat.
Compositions
6.5
6.0
5.5
10.5
5.0
10.0
15.0
9.5
14.5
19.0
18.5
22.5
C18 H34 N10 O7 Cl1
C20 H36 N7 O8 Cl1
C22 H38 N4 O9 Cl1
C23 H34 N8 O5 Cl1
C24 H40 N1 O10 Cl1
C25 H36 N5 O6 Cl1
C26 H32 N9 O2 Cl1
C27 H38 N2 O7 Cl1
C28 H34 N6 O3 Cl1
C32 H32 N5 O1 Cl1
C34 H34 N2 O2 Cl1
C39 H34 N0 O0 Cl1
Not
enough
C!
Too many
N or O
Not
enough N
or O or too
many C
Technology Transition Workshop
Elemental Composition Calculation
12/12/2007
11:25:13 AM
Element Limits:
C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1
Tolerance: 5.00 mmu
Even or odd electron ion or both: BOTH
Minimum unsaturation: -0.5
Maximum unsaturation: 100.0
Meas. mass
u
537.233582
From 2003 PDR:
Hydroxyzine empirical formula
C21H27N2O2Cl
OK. NOW WHAT??
Abund. Diff.
%
mmu
5.76
-3.19
-4.50
4.05
Unsat.
Compositions
9.5
14.5
19.0
C27 H38 N2 O7 Cl1
C28 H34 N6 O3 Cl1 Too many
nitrogens!
C32 H32 N5 O1 Cl1
Vistaril (hydroxyzine pamoate)
Sucrose is C12H22O11
Inert ingredients:
Magnesium stearate
Sodium lauryl sulfate
Starch
Sucrose
LOOK HERE!!
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Sucrose peaks!
Sucrose – ori1=20V
289.0942
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Hydroxyzine empirical formula:
C21H27N2O2Cl
Target compound:
C27H38N2O7Cl
BEST GUESS:
C21H27N2O2Cl + C6H12O6 = C27H39N2O8Cl (not quite!)
Hydrolyzed sucrose
But, if subtract H2O:
C27H37N2O7Cl (Still off by one!)
Ionized to:
[C27H37N2O7Cl + H]+
=
537.2367 Da (calculated)
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MIXTURES
Magically disappearing ions!
Codeine std
ori 1 30V
Codeine:APAP 20:1
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MIXTURES
Codeine:APAP 10:1
Codeine:APAP 5:1
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MIXTURES
Codeine:APAP 2:1
Codeine:APAP 1:1
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“Today
is
done!”