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Transcript of Determination of Analysis by LC-MS/MS Endogenous … · Endogenous Steroids in Biomatrices Analysis...
Determination of
Endogenous Steroids in
Biomatrices
Analysis by LC-MS/MS
for Clinical Research
Andre Szczesniewski
Senior Applications Chemist
Agilent Technologies
November 29, 2012
November 29, 2012 1
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 2
Overview
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 3
• Sensitivity
• Detection limits down to picomolar levels
• Specificity
• Compound specific response reduces false positives or negatives
• Selectivity
• Differentiate interferences from matrix, background and other noise
• Quantitation
• Wide linear dynamic range and accurate quantitation
• Throughput
• Short analysis time and multiple analytes in a single test
Why Labs Are Interested in LC/MS?
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 4
Cholesterol Metabolism Steroidogenesis
For Research Use Only. Not for use in diagnostic procedures.
What is Multiple Reaction Monitoring (MRM)?
November 29, 2012 5
170 210 250 290
210
222
268 280 165
Spectrum with
background
ions (from ESI)
Q1 lets only
target ion 210
pass through
190 210
210
Collision cell
breaks ion 210
apart
150 170 190 210
210 158
191
Q3 monitors only
characteristic
fragment 158
from ion 210 for
quantitation
160
158
no chemical background
Ions
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 6
For Research Use Only. Not for use in diagnostic procedures.
Method Development Objectives
November 29, 2012 7
• Comprehensive, quantitative method for the
analysis of major cholesterol steroidal metabolites
for use in the clinical research laboratories
• Simple and economical sample preparation
• Robust and reliable workflow
• Easily modifiable to accommodate and adjust
changing research requirements
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 8
Cholesterol
Pregnenolone Progesterone Deoxycorticosterone Corticosterone
Aldosterone
17α-OH Pregnenolone 17α-OH Progesterone Cortisol
Testosterone
Dihydrotestosterone
Cortisone
Estrone Estradiol Dehydroepiandrosterone
Androstenediol
11-Deoxycortisol
Androstenedione
Estriol
Cholesterol Steroidogenesis Analysis of 13 Steroids
For Research Use Only. Not for use in diagnostic procedures.
Structures and Molecular Weights Steroidogenesis
November 29, 2012 9
Cortisone
C21H28O5
M.W. 360.2
Aldosterone
C21H28O5
M.W. 360.2
11-Deoxycortisol
C21H30O4
M.W. 346.2
Corticosterone
C21H30O4
M.W. 346.2
Cortisol
C21H30O5
M.W. 362.46
Androstenedion
e
C19H26O2
M.W. 286.2
DHEA
C19H28O2
M.W. 288.2
17-Hydroxyprogesterone
C21H30O3
M.W. 330.2
Dihydortestosterone
C19H30O2
M.W. 290.2
Progesterone
C21H30O2
M.W. 314.2
Pregnenolone
C21H32O2
M.W. 316.2
17-OH-Pregnenolone
C21H32O3
M.W. 332.2
Testosterone
C19H28O2
M.W. 288.2
5-delta Steroids
0
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 10
For Research Use Only. Not for use in diagnostic procedures.
Sample Preparation Liquid-Liquid Extraction
November 29, 2012 11
500 μL of plasma or serum + 100 μL ISTD at 5ng/ml
Vortex 30 seconds
Add 5 mL MTBE*
Vortex/mix 5 minutes
Transfer MTBE layer (top) to a test tube
then evaporate under N2 at 35 °C
Reconstitute in 100 μL in 30% Methanol
Analyze by LC-MS/MS
* Methyl tert-butyl ether For Research Use Only. Not for use in diagnostic procedures.
Method Parameters 1290 UHPLC System
November 29, 2012 12
Injection volume: 10 mL
Autosampler temp: 5 ºC
Column Temp: 50 ºC
Needle Wash: 50%IPA : 25% MeOH : 25% H2O
Column: Poroshell 120 EC-C18, 2.1x50 mm, 2.7 mm
Pump Flow: 0.4 mL/min
Mobile Phase: A = Water + 5mM NH4FA
B = Methanol + 5mM NH4FA
Gradient: Time %B
0.00 10
0.50 10
8.00 40
9.00 60
9.50 60
9.60 95
10.4 95
10.5 10
Stop time – 10.5 min
Post time – 1.00 min For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 13
6460 QQQ MS conditions
Ion Mode: Jetstream, Positive
ESI conditions
Drying Gas Temperature: 250 ºC
Drying Gas Flow: 11 L/min
Nebulizer Pressure: 35 psi
Sheath Gas Temperature: 350 ºC
Sheath Gas Flow: 11 L/min
Capillary: 3000 V
Nozzle Voltage: 0 V
EMV: 300 V
Resolution: MS1 - Unit, MS2 – Unit
Dwell Time 20 ms
Cell Acceleration 3 V
• The selection of the spray chamber conditions for a multi-component method always involves
a trade-off between the optimum settings of the individual compounds.
Method Parameters 6460 QQQ System
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 14
Method Parameters MRM Transition Parameters for Analytes
Comound Name Precursor Ion Product Ion Quant-Qual
Frag (V) CE (V) Quant-Qual
11-Doxycortisol 347.2 109.1 - 97.1 95 28 - 28
17a-Hydroxypregnenolone 315.2 297.1 - 255.1 93 2 - 7
17a-Hydroxyprogesterone 331.2 109.1 - 97.1 100 27 - 27
Aldosterone 361.2 343.2 - 315.2 100 15 - 16
Androstenedione 287.2 109.1 - 97.1 100 25 - 17
Corticosterone 347.2 329.2 - 121.1 110 12 - 24
Cortisol 363.2 121.1 - 91.1 105 24 - 60
Cortisone 361.2 163.1 - 121.1 120 20 - 32
DHEA 289.2 271.2 - 231.2 95 2 - 20
Dihydrotestosterone 291.2 255.2 - 91.1 120 12 - 52
Pregnenolone 317.2 299.2 - 159.1 102 4 - 16
Progesterone 315.2 109.1 - 97.1 100 25 - 20
Testosterone 289.2 109.1 - 97.1 100 25 - 25
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 15
Comound Name Precursor Ion Product Ion Frag (V) CE (V) 11-Doxycortisol-D5 352.2 100.1 95 28
17a-Hydroxypregnenolone-13C2-D2 319.2 301.1 93 2
17a-Hydroxyprogesterone-D8 339.2 100.1 100 27
Aldosterone-D8 369.2 351.2 100 15
Androstenedione-3C13 290.2 100.1 100 17
Corticosterone-D8 355.2 337.2 110 12
Cortisol-D4 367.2 121.1 105 24
Cortisone-D7 369.2 169.1 120 20
DHEA-D5 294.2 258.2 95 2
Dihydrotestosterone-D3 294.2 258.2 120 12
Pregnenolone-13C2-D2 321.2 303.2 102 4
Progesterone-D9 324.2 100.1 100 20
Testosterone-D3 292.2 97.1 100 25
Method Parameters MRM Transition Parameters for Internal Standards
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 16
For Research Use Only. Not for use in diagnostic procedures.
Chromatography 13 Steroid Panel
November 29, 2012 17
Co
rtis
ol
Ald
os
tero
ne
Co
rtis
on
e
Co
rtic
oste
ron
e
11
-De
ox
yc
ort
iso
l
17a-
Hyd
rox
yp
rog
es
tero
ne
Pre
gn
en
olo
ne
17
-Hyd
rox
yp
reg
nen
olo
ne
Pro
ge
ste
ron
e
Dih
yd
rote
sto
ste
ron
e
DH
EA
Te
sto
ste
ron
e
An
dro
ste
ne
dio
ne
5-delta Steroids
For Research Use Only. Not for use in diagnostic procedures.
Chromatography Isobaric Pairs
November 29, 2012 18
Ald
os
tero
ne
Co
rtis
on
e
Co
rtic
os
tero
ne
11-D
eo
xyc
ort
iso
l
Te
sto
ste
ron
e
DH
EA
For Research Use Only. Not for use in diagnostic procedures.
• Poroshell 120 Columns have:
• 80-90% efficiency of sub 2um
• At ~40-50% lower pressure
• 2X efficiency of 3.5um (totally porous)
• A 2um frit to reduce clogging
• A 600 bar pressure limit for HPLC or
UHPLC use
• The superficially porous particle is 2.7um with
a solid core (1.7um) and porous outer layer
with a 0.5um diffusion path
19
Poroshell 120 is a high efficiency, high resolution column choice for
enhancing productivity in LC and LC/MS
1.7um
0.5um
0.5um
Poroshell 120 Columns For HPLC and UHPLC
For Research Use Only. Not for use in diagnostic procedures.
Diflusinal in Plasma
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
1 501 1001 1501 2001 2501
Injections
Pre
ssu
re
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Eff
icie
ncy (
N)
End Press
Plates
Column: Poroshell 120 EC-C18, 3.0 x 50mm, 2.7um LC: Agilent 1200 RRLC (SL)
Sample: Precipitated Plasma: 2 parts Plasma: 7 Parts 20/80 Water-MeCN w/0.1 % Formic Acid with 1 Part Diflusinal
in 50/50 Water-MeCN 10 ug/ml (Final concentration Diflusinal 1 ug/ml) Shaken and allowed to settle 10 minutes
Not Centrifuged/ Not Filtered Injection Volume: 1ul injections
Solvent A: Water w/0.1 % TFA
Solvent B: MeCN w/0.08 % TFA
Flow Rate 1 ml/min 1 ul injection
Time % B
0 20
0.5 90
0.6 90
1.1 20
2.5 20
Good lifetime with
plasma samples!
20
Poroshell 120 Resists Plugging with 2µm Frit Use with Plasma or Similar Matrices
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 21
Aldosterone
Cortisol Testosterone
Calibration Curves Representative Steroids
17-Hydroxypregnenolone
For Research Use Only. Not for use in diagnostic procedures.
Results Summary of Analytical Performance
22
Compound R2 Level Conc.
Compound R2 Level Conc.
(pg/mL) (pg/mL)
Aldosterone 0.998
LOD 5
DHEA 0.995
LOD 250
LLOQ 10 LLOQ 500
ULOQ 500000 ULOQ 100000
Cortisone 0.998
LOD 5
17α-Hydroxyprogesterone 0.996
LOD 10
LLOQ 10 LLOQ 25
ULOQ 1000000 ULOQ 100000
Cortisol 0.999
LOD 1
17-Hydroxypregnenolone 0.995
LOD 250
LLOQ 5 LLOQ 500
ULOQ 1000000 ULOQ 250000
Corticosterone 0.996 LOD 5
Dihydrotestosterone 0.997 LOD 5
LLOQ 10 LLOQ 10
ULOQ 100000 ULOQ 500000
11-Deoxycortisol 0.995
LOD 2.5
Progesterone 0.995
LOD 25
LLOQ 5 LLOQ 50
ULOQ 500000 ULOQ 500000
Androstenedione 0.999
LOD 2.5
Pregnenolone 0.999
LOD 1000
LLOQ 5 LLOQ 2500
ULOQ 1000000 ULOQ 500000
Testosterone 0.996
LOD 5
LLOQ 10
ULOQ 500000 For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 23
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 25
For Research Use Only. Not for use in diagnostic procedures.
Method Development Objectives
November 29, 2012 26
• Simple, quantitative method for the analysis of 17ß-
Estradiol (E2) and Estrone (E1) for use in clinical
research laboratories
• Reliable and solid workflow
• Utilize state of the art LC/MS instrumentation with
superior sensitivity to meet challenging limits of
detection
For Research Use Only. Not for use in diagnostic procedures.
Cholesterol Steroidogenesis Analysis of Estrogens
November 29, 2012 27
Cholesterol
Pregnenolone Progesterone Deoxycorticosterone Corticosterone
Aldosterone
17α-OH Pregnenolone 17α-OH Progesterone Cortisol
Testosterone
Dihydrotestosterone
Cortisone
Estrone Estradiol Dehydroepiandrosterone
Androstenediol
11-Deoxycortisol
Androstenedione
Estriol
For Research Use Only. Not for use in diagnostic procedures.
Estrogens Estradiol and Estrone
November 29, 2012 28
17ß-Estradiol
C18H24O2
M.W. 272.2
Estrone
C18H22O2
M.W. 270.2
For Research Use Only. Not for use in diagnostic procedures.
17ß-Estradiol Dealing with a Difficult Analysis
Analytical Challenge
• Low level detection of a compound lacking of highly ionizable
functional groups
Current Approaches
• Immunoaffinity Extraction
• Solid-Phase Extraction
Drawbacks
• Costly
• Time-Consuming
• Introduce Variability
November 29, 2012 29
OHCH3
H
H
H
OH
For Research Use Only. Not for use in diagnostic procedures.
Increased Ion Generation Agilent JetStream Technology
November 29, 2012 30
•Thermal confinement of
nebulizer spray
•Efficient desolvation to
create gas phase ions
•Creates an ion rich zone
For Research Use Only. Not for use in diagnostic procedures.
Increased Sensitivity Using Ion Funnel Technology Agilent 6490 QQQ
November 29, 2012 31
Hexabore for
greater
sampling
Removal of
carrier gas
and neutrals
Higher ion
yield
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 32
For Research Use Only. Not for use in diagnostic procedures.
Sample Preparation Liquid-Liquid Extraction (LLE) and Derivatization
November 29, 2012 33
500 µL of serum + ISTD
Vortex 20 sec, let it stand 5 minutes in room temp.
Add 50 µL of 100 mM Sodium Biocarbonate at pH 10.5
and 50 µL of 1mg/mL Dansyl Chloride
Incubate at 60ºC for 5 minutes
Add 6 mL of 3:2 of Hexane:Dichloromethane, vortex 60
sec then centriufuge at 3000 rpm for 10 min
Collect organic layer (top), dry down with N2 at 40ºC
Add 100 µL of water and analyze by LC-MS/MS
For Research Use Only. Not for use in diagnostic procedures.
Estradiol Derivatization Dansyl Chloride
November 29, 2012 34
SO O
NCH3 CH3
CH3OH
H
H H
OOHCH3
H
H
H
OH
Estradiol Dansyl Chloride
+
Dansyl-Estradiol
For Research Use Only. Not for use in diagnostic procedures.
On-Sample Cleanup System Lab Bench Layout
November 29, 2012 35
1: Loading pump
2: Analytical pump
3: Autosampler
4: Column compartment
1
3
4
2
For Research Use Only. Not for use in diagnostic procedures.
Online Sample Cleanup System Diagram Forward Flush
November 29, 2012 36
Trapping Column
Waste
Loading
Pump
(1260)
+ALS
QQQ MS
Analytical Column
Analytical
Pump
(1290)
For Research Use Only. Not for use in diagnostic procedures.
Online Sample Cleanup Operation Loading, Cleaning, Transferring and Analyzing
November 29, 2012 37
Trapping
Column
Waste
Loading
Pump
(1260)
+ALS
QQQ MS
Transfer Position
Analytical
Column
Analytical
Pump
(1290)
Trapping
Column
Waste
Loading
Pump
(1260)
+ALS
QQQ MS
Loading/Analysis Position
Analytical
Pump
(1290)
Analytical
Column
For Research Use Only. Not for use in diagnostic procedures.
Injection volume 40 µL
Needle Wash flushport (methanol:water 75:25), 5 seconds
Injector Temp 4 °C
Columns Loading: Zorbax XDB-C18, 2.1 x 30mm, 3.5 µm
Analysis: Poroshell EC-18, 2.1 x 50 mm, 2.7 µm
Column Temp 50 °C
Switching Valve 0.0 min Pos1
1.9 min Pos 2
2.4 min Pos 1
Stop Time 7.7 minutes
Post Time 0.3 minute
Mobile Phase Chanel A = H2O + 5mM Ammonium Formate
Chanel B = Methanol + 5mM Ammonium Formate
Pump Gradients
Pump 1 Pump 2
LC Parameters 1290 HPLC With Online Sample Cleanup
November 29, 2012 38
Time Flow % Solvent B
0.0 0.3 50
2.0 0.3 70
4.0 0.3 75
6.2 0.3 78
6.3 0.6 98
7.5 0.6 98
7.6 0.6 50
7.7 0.3 50
Time Flow % Solvent B
0.0 0.5 50
2.0 0.5 70
5.0 0.5 75
5.1 0.5 98
7.5 0.5 98
7.6 0.5 50
For Research Use Only. Not for use in diagnostic procedures.
Polarity Positive
Drying Gas Temp 150⁰C
Drying Gas Flow 17 L/min
Nebulizer Pressure 45 psi
Sheath Gas Temp 400 °C
Sheath Gas Flow 12 L/min
Capillary Voltage 4500 V
Nozzle Voltage 500V
MS Method Parameters 6490 QQQ System
November 29, 2012 39
Compound Precursor Product Frag (V) Dwell (msec) CE (V) Cell Acc Estradiol 506.3 171.1 380 100 41 3 Estradiol-d5 511.3 171.1 380 100 41 3 Estrone 504.3 171.1 380 100 35 3 Estrone-d4 508.3 171.1 380 100 35 3
For Research Use Only. Not for use in diagnostic procedures.
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
November 29, 2012 40
For Research Use Only. Not for use in diagnostic procedures.
Derivatized Estradiol Extracted From Serum LLOQ 0.25pg/mL
November 29, 2012 41
Injection Area
1 5323
2 4568
3 4574
4 4277
5 5522
6 5237
7 5444
8 5483
9 4615
Average 5004
SD 487
CV (%) 9.7
LOQ: 0.25 pg/mL
10fg on column
Blank
For Research Use Only. Not for use in diagnostic procedures.
Estradiol Calibration Curve 0.25-100 pg/mL
November 29, 2012 42
Concentration (pg/ml)
0 10 20 30 40 50 60 70 80 90 100
x10-1
0
1
2
3
4 y = 0.004 * x + 3.83E-004
R^2 = 0.999
Re
lative
Re
sp
on
se
0 0.2 0.4 0.6 0.8 1.0
x10-3
0
1
2
3
4
Level Expected Average Accuracy (%) SD CV (%)
1 0.25 0.25 82 - 115 0.02 8.45
2 0.50 0.49 81 - 114 0.04 9.01
3 1.00 0.97 88 - 112 0.07 6.81 n = 15; all measurements in pg/ml
For Research Use Only. Not for use in diagnostic procedures.
Derivatized Estrone Extracted From Serum LLOQ 0.25pg/mL
November 29, 2012 43
Injection Area
1 4207
2 4272
3 3703
4 3822
5 3724
6 3842
7 4184
8 4256
9 3879
Average 3988
SD 237
CV (%) 6.0
LOQ: 0.25 pg/mL
10fg on column
Blank
For Research Use Only. Not for use in diagnostic procedures.
Estrone Calibration Curve 0.25-100 pg/mL
November 29, 2012 44
Level Expected Average Accuracy (%) SD CV (%)
1 0.25 0.27 99 - 121 0.02 6.11
2 0.50 0.50 91 - 110 0.03 5.00
3 1.00 0.92 86 - 101 0.04 4.50 n = 15; all measurements in pg/ml
0.0 0.2 0.4 0.6 0.8 1.0
x10-3
0
1
2
3
4
5
Concentration (ng/ml)
0 10 20 30 40 50 60 70 80 90 100
x10-1
0
1
2
3
4
y = 0.004 * x - 1.146E-004
R^2 = 0.997
Re
lative
Re
sp
on
se
For Research Use Only. Not for use in diagnostic procedures.
November 29, 2012 45
• Introduction
• 13 Steroids Panel Analysis
• Sample Preparation & Methodology
• Results
• Questions Break
• Estradiol and Estrone Analysis
• Sample Preparation and Methodology
• Results
• Summary
• Questions
For Research Use Only. Not for use in diagnostic procedures.
Conclusions
November 29, 2012 46
• Comprehensive method for steroids profile and a highly sensitive
and robust method for low-level quantitation of Estradiol and Estrone
has been developed
• Sample preparation consists of a straightforward LLE and addition of
derivatization for estrogens
• Steroid Panel LLOQs were in the low pg/ml range while 5-Δ steroids
were in high pg/mL. Dynamic range of 4 orders of magnitude with
R2 ≥ 0.995
• LLOQ of 0.25 pg/ml has been achieved for derivatized Estradiol and
Estrone with an R2 ≥ 0.995
• Number of steroids and analysis time can be reduced to according to
research needs.For Research Use Only. Not for use in diagnostic procedures.
Tips and Tricks
November 29, 2012 47
• Mobile phase quality; grade and freshness
• Inline filter between needle seat and injection valve
• Make internal standards in deuterated methanol
• If sensitivity decreases, check cleanness of a spray chamber
and run MS2 scan method to monitor background ions levels
• Divert the flow to waste for the first minute to remove potential
residual salts or unreacted derivatization reagent
• Use needle wash to reduce carryover
• Keep the column compartment closed (cover on) to avoid
variations due to external temperature fluctuations
For Research Use Only. Not for use in diagnostic procedures.
List of Columns and Supplies
November 29, 2012 48
Supplier Part No. Description
Agilent 695775-902 Analytical column: Poroshell 120 EC-C18,
2.1x100 mm, 2.7 µM (Estrogens)
Agilent 820555-901 Trapping column hardware kit (Estrogens)
Agilent 973700-932 Trapping Column: Zorbax Eclipse XDB-C18,
2.1x 30 mm, 3.5 µM (Estrogens)
Agilent 699775-902 Analytical column: Poroshell 120 EC-C18,
2.1x50 mm, 2.7 µM (Panel)
Agilent 5067-4638 1290 Infinity Inline filter, 0.3 µM
Agilent 5182-0556 2.0 mL Amber vials and caps
For Research Use Only. Not for use in diagnostic procedures.
List of Reagents
November 29, 2012 49
Biological Solutions
Human Serum Double Charcoal Stripped, Delipidized Golden West Biologicals
Bovine Serum Albumin Golden West Biologicals
Solvents
Methanol Honeywell
Water Millipore
MTBE Honeywell
Hexane Honnewell
Dichloromethane Honeywell
Other Chemicals
Ammonium Formate Fisher
Dansyl Chloride Sigma-Aldrich
Sodium Bicarbonate Sigma-Aldrich
Standards and Internal Standards Cerillinat, IsoSciences
For Research Use Only. Not for use in diagnostic procedures.