AN EVALUATION OF EXTRACTION PARAMETERS AND LCMS ANALYSIS OF OLIGONUCLEOTIDES FROM BIOLOGICAL MATRICES JENNIFER ELAINE FELTS A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Department of Chemistry and Biochemistry University of North Carolina Wilmington 2008 Approved by Advisory Committee ______________________________ ______________________________ Dr. Bruce Petersen Dr. John Tyrell ______________________________ Dr. James Reeves, Chair Accepted by ______________________________ Dr. Robert D. Roer Dean, Graduate School
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AN EVALUATION OF EXTRACTION PARAMETERS AND LCMS ANALYSIS OF OLIGONUCLEOTIDES FROM BIOLOGICAL MATRICES
JENNIFER ELAINE FELTS
A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of Master of Science
Department of Chemistry and Biochemistry
University of North Carolina Wilmington
2008
Approved by
Advisory Committee
______________________________ ______________________________ Dr. Bruce Petersen Dr. John Tyrell
______________________________
Dr. James Reeves, Chair
Accepted by
______________________________ Dr. Robert D. Roer
Dean, Graduate School
Table of Contents Page
Abstract .............................................................................................................................. iv
Oligonucleotide synthesis is done via a cycle of four chemical reactions that are repeated until all desired bases have been added: • Step 1 - De-blocking (detritylation): The dimethoxyltrityl is removed with an acid, such as trichloroacetic acid, and washed out, resulting in a free 5' hydroxyl group on the first base. • Step 2 - Base condensation (coupling): A phosphoramidite nucleotide (or a mix) is activated by tetrazole which removes the iPr2N group on the phosphate group. After addition, the deprotected 5' OH of the first base and the phosphate of the second base react to join the two bases together in a phosphite linkage. These reactions are not done in water but in tetrahydrofuran or in dimethyl sulfoxide. Unbound base and by-products are washed out. • Step 3 - Capping: About 1% of the 5' OH groups do not react with the new base and need to be blocked from further reaction to prevent the synthesis of oligonucleotides with an internal base deletion. This is done by adding a protective group in the form of acetic anhydride and 1-methylimidazole which react with the free 5' OH groups via acetylation. Excess reagents are washed out. • Step 4 - Oxidation: The phosphite linkage between the first and second base needs to be stabilized by making the phosphorous pentavalent. This is achieved by adding iodine and water which leads to the oxidation of the phosphite into phosphate. This step can be substituted with a sulphorylation step for thiophosphate nucleotides.
Figure 2: Oligonucleotide Synthesis, cont. 6
11
Figure 3: Antisense Oligonucleotides in Action7
12
DNA/RNA hybrid can be degraded by the enzyme RNase H.”8 “The best-characterized
antisense mechanism results in cleavage of the targeted RNA by endogenous cellular
nucleas
s
the desired speed and yield of the analysis, due
to the l
e
C and
t.
s a
c
es, such as RNase H or the nuclease associated with the RNA interference
mechanism. However, oligonucleotides that inhibit expression of the target gene by
noncatalytic mechanisms, such as modulation of splicing or translation arrest, can also be
potent and selective modulators of gene function.”7 As the list of bioanalytical uses grow
longer, the need for quantifiable analytical and purification techniques grows more
compelling.
II. History of Oligonucleotide Analysis
The introduction of tandem electrospray ionization (ESI) and mass spectroscopy
(MS/MS) analysis has greatly improved the analytical detection of these polymers. The
previously used technologies, typically polyacrylamide gel electrophoresis (PAGE),
capillary gel electrophoresis (CGE), or anion-exchange high pressure liquid
chromatography (HPLC) techniques, lack
ong analysis and/or instrumentation time required.4 Gel electrophoresis is a
technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein
molecules using an electric current applied to a gel matrix, while HPLC uses the
separation science of mobile phase loading on an analytical column and resulting analyt
retention times to identify different compounds.9
While the liquid and column chromatography remain constant between HPL
HPLC/MS/MS, the mode of detection between the two analytical tools are very differen
HPLC uses a photodiode array detector to monitor peak elution, while MS/MS utilize
“mass analyzer, which sorts the ions by their masses by applying electric and magneti
13
fields, and a detector, which measures the value of some indicator quantity and thus
provides data for calculating the abundances each ion fragment present.”10 “Mass
spectrometry is an analytical technique that identifies the chemical composition of a
e mass-to-charge ratio of charged particles.”10 This
techniq utilizes
nd solvent
air reverse
sue
t
inued research, as this analytical technique has the capacity
compound or sample on the basis of th
ue offers improved limits of detection with reduced injection times, and
ESI to produce charged ions for sampling from a volatile spray of analyte a
forced through a charged ions for sampling from a volatile spray of analyte and solvent
forced through a charged capillary tube.
However, with the advances of the currently touted technique of ion-p
phase HPLC/MS/MS (IP-RP HPLC/MS/MS) come new difficulties. These difficulties are
chiefly, a) the low response of these polymers on the mass spectrometer, b) the
prevalence of alkali (Na+, K+) cation adduction, c) chromatographic complexity due to
the multiple charge state peaks of these polymers, and d) the intricacy of HPLC
separation of oligonucleotides, which can be dependent on both the length and the
composition of the polymer.11 In addition, the analysis of these compounds from tis
cells can result in unclean extracts unfavorable to the attainment of desired detection
limits, typically nanograms of analyte per gram tissue sample. However, this pursui
shows great promise with cont
for on-line desalting, oligonucleotide chromatographic separation and characterization,
among other possibilities.
III. Chromatographic Considerations
The investigation into improved separation techniques requires further
understanding of the scientific processes at work during chromatography.
14
Chromatography in its simplest form is the separation of a mixture based on its chem
characteristics. This mixture is moved through a stationary phase, which selectively
retains components of the mixture for further analysis or measure. “Column
chromatography is a separation technique in which the stationary bed is within a tube.
The particles of the solid stationary phase or the support coated with a liquid stationary
phase may fill the whole inside volume of the tube (packed column) or be concentrated
on or along the inside tube wall leaving an open, unrestricted path for th
ical
e mobile phase in
ferences in rates of movement through
the me
e
of
improving
the middle of the tube (open tubular column). Dif
dium are reflected in different retention times of the sample.”12 The binding
affinities of the compounds are affected by a variety of intermolecular forces, such as the
ionic strength and polarity, which can then be channeled into a desired function of
chromatographic separation.
The essential function in this chromatographic separation is performed by ion-exchang
chromatography. This form of separation science uses the charge properties of the
compounds and stationary phase to selectively retain the desired components while
flushing unwanted molecules through the system. The choice of stationary phase sorbent
is determined by the characteristic ionic functional groups displayed; these groups
interact with analyte ions of opposite charge.13 As there can be both positively and
negatively charged functional groups on oligonucleotides, their overall net charge, and
strength of retention, can be influenced by mobile phase composition. As the
concentration of similarly charged species in the mobile phase is increased, the binding
the analytes to the stationary phase will decrease, and elution will occur.13 By
15
our understanding of the interactions governing analyte retention on the stationary phase,
a more controlled separation of the target compound can be obtained.
IV. Guidelines for Intended Assay Improvement
Through the work of Martin Gilar, Kenneth J. Fountain, and numerous other
researchers, several important and reproducible parameters for IP-RP-HPLC
have surfaced.3,4,15,16,18,19 Oligonucleotides are best analyzed in negative ion mode,
ion-pairing reagents in the mobile phases.15 To remove alkali and DNAase/RNAase
molecules, the use of distilled water or Water for Injection (WFI) water is preferred.4
Cation adducts can be reduced by the addition of ammonium ions, which should be
administered at a near-neutral pH to maintain MS response.4 Chromatography is greatly
improved with the addition of a column heater set at 60 °C and the use of smaller micron
pore size.16 The oligonucleotide will undergo fragmentation by 1,2 elimination of nu
bases at either the 5’ or the 3’ side of the nucleic sugar.1 This fragmentation yields
metabolites termed N-(x, number of nucleic bases lost), which consist of the original
oligonucleotide minus one or more nucleic bases. Each metabolite can have two forms,
depending on whether the bases were removed from the 5’ or 3’ position of the sugar,
and the N-1 metabolite forms are typically identical when analyzed by mass
spectrometer. Separation of the oligomers can be improved with lower flow rates, and the
separation increases in difficulty as the oligomer increases in length.16 These results have
demonstrated repeated occurrence through the work of many researchers.1,3,4,15,16,17,18,1
By investigating similar polymers and conditions, researchers have reached a variety o
conclusions regarding optimization. Several mobile phase components have been studi
including triethylamine (TEA), 1,1,1,3,3,3-Hexafluoro-2-propanol (H
v/v; 1:10 DEA(diethylamine)/HFIP, v/v; 25 mM imidazole/piperidine, pH=8; 1:10
TEA/HFIP, v/v, pH=4; and 1:10 TEA/HFIP, v/v, pH=6. All of the mobile phase in-
pairing reagents were obtained from Sigma, while the WFITM water was purchased
through Hyclone and the methanol from Burdick & Jackson.
IV. Analytical Column
Each column was conditioned with the ion-pairing mobile phases, and the
for three injections of a
The columns tested for this experiment were Waters Xterra MS C18, 50 x 2.1 mm, 2.5
µm (Part No. 186000594); Grace Division Discovery Genesis C8, 50 x 2.1 mm, 3 µm
(Part No. FK5968EJ); Thermo-Scientific Hypersil Gold, 50 x 2.1 mm, 3µm (Part No.
25003-052130); Thermo-Scientific Betamax Base, 50 x 2.1 mm, 5µm (Part No. 95105-
052130); Thermo-Scientific Aquasil C18, 50 x 2.1 mm, 3 µm (Part No. 77503-052130);
Phenomenex Synergi Max-RP, 30 x 2 mm, 4 µm (Part No. 00B
Synergi Pola
23
50
mm, 3
V. E
e
out either the reconstitution solution nor
the
duplica t
fort
set a 90:10:1 WFITM water/methanol/TEA solution. In addition,
another set of t n
above and stor
VI. SPE Invest
SPE experimen
The 00 mM HFIP solution
prior to
better enhance sorbent binding of the analyte. Blanks and two levels of QCs (1.0 and
oth plasma and liver tissue, using
rocedure:
1. Transfer a 0.100-mL aliquot of blank bulk matrix lysate-homogenate or plasma to 20 µL of the working solution (all but post samples,
groups 3,4) into the homogenate tube. Add 20 µL of IS1 (all but post samples, ppropriate.
2. Add 200 µL of WFI water to each sample. 3. 4. Add 300 µL of 25:24:1 phenol/chloroform/isoamyl alcohol to each sample.
x 2 mm, 5 µm (Part No. A3040050x020); and the Phenomenex Phenyl-hexyl, 50 x 2
µm (Part No. 00B-4256-B0).
xtraction Details
A number of small extraction tests were performed. The extraction procedur
lined in Appendix C was followed. However, n
storage conditions of the final extracts had been tested. To test these conditions,
te samples were extracted from porcine liver homogenate using the parameters se
h in Appendix C, with one set being reconstituted in WFITM water (step 13) and one
being reconstituted in
he ion-pairing reconstitution condition samples was extracted as writte
ed in silylated glass inserts (rather than plastic) prior to injection.
igation
tation continued with further testing of various solid-phase techniques.
HLB bed was conditioned with acetonitrile and a 8mM TEA/ 1
loading. In addition, ion-pairing reagent was added to each sample prior loading
to
400 ng/mL and ng/g, respectively) were extracted in b
the following method. The samples were extracted as follows:
P
a Phase Lock Gel tube. Add
groups 3,4) as a
Add 100 µL of concentrated ammonium hydroxide to each sample.
5. Gently rock each tube several times and then mix vigorously for thirty seconds.
24
6. Centrifuge at 15000 rpm for five minutes.
8. Gently rock each tube several times, mix vigorously for thirty seconds, and
9. Add 400 µL of 8 mM TEA/100 mM HFIP in water to Group 1 samples and 100
10. Transfer the aqueous extract the appropriate SPE filtration tube during the lo
11. SPE extraction:
1.0 mL 8 mM TEA/100 mM HFIP in water. Load the sample slowly.
100 mM TEAA. Elute with 1.0 mL 60:40 ACN/8 mM TEA, v/v.
TEA/WFI Water/Methanol, v/v/v, 1.0 mL methanol, 1.0 mL water and 1.0
TEAA, and 1.0 mL water. Elute with 1.0 mL 90:10 Methanol/Water, v/v.
13. Evaporate under
7. Add 300 µL of chloroform to each sample.
centrifuge at 15000 rpm for five minutes.
µL of 1M TEAA to Group 2 samples. ad
step.
Groups 1, 3: Condition an HLB 10 mg cartridge with 1.0 mL ACN, and
Wash with 0.300 mL 8 mM TEA/100 mM HFIP in water, and 0.500 mL
Group 2, 4, 5: Condition an HLB 10 mg cartridge 1.0 mL of 1:10:90
mL 0.1 M TEAA. Load the sample slowly. Wash with 1.0 mL 0.1 M
12. Spike 20 µL of analyte and IS spiking solutions into Group 3 and 4 samples. a nitrogen stream at approximately 45 °C.
14. Reconstitute with 100 µL of WFI water. Transfer extracts to a plastic well in a 96-well plate. Seal the plate and mix vigorously for 30 sec, then centrifuge at 3500
minutes. Group Group GroGro tracted as Gro As covery, the load and wash eluents
wer
VII
previous SPE procedures tested had not yet been optimized, further
investigation into t
to examine the most fu tissue
samples, and held in c acted without the addition of SPE.
These non-SPE extrac , and
transferring the extracts from the Phase-Lock gel tubes (Eppendorf) in step 7 to the
There is no initial retention of the majority of the analyte, resulting in jagged band elution.
42
Figure
11: 1:10:500 TPA(tripropylamine)/HFIP/MP, v/v/v
There is no initial retention of the compound, leading to an analyte response in the
solvent front.
43
Figure 12: 1:10:500 DEA/HFIP/MP, v/v/v
There is limited initial retention of analyte, resulting in an analyte response in the solvent front and lost response at the expected retention time.
44
Further testing was conducted on the 1:10:500 TEA/HFIP/Solvent, v/v/v, mobile
phases, within a wider range of pH. The mobile phase has a natural pH of ~8, which is
the preferred range for these compounds. Indeed, during further pH testing, no analyte
peaks were observed at pH=4, indicating that the compounds cannot be eluted or
measured under acidic conditions, most likely due to the binding of the highly protonated
TEA molecule (Figure 14). When the pH of the mobile phases was raised to 6, analyte
peaks were observed (Figure 15). However, these peaks exhibited poor chromatography,
indicating poor banding and incomplete elution from the column. Clearly, the TEA
cannot be too heavily protonated during oligonucleotide chromatography, as the clean
banding elution cannot be readily achieved in this manner. There was initial interest in
adjusting the pH of the assay to increase the prevalence of a concentrated number of
mass-to-charge states; however, as mobile phase pH adjustments were found to be
detrimental, this projected avenue could not be pursued. Additional modifications of the
ass spectroscopy parameters, such as collision energy and ion source temperature, were
lso found to be unproductive, as the parameters optimized during tuning for the
ompounds adhered to a narrow set of conditions.
From this testing of mobile phases components and conditions, a final mobile
hase set was determined to be 1:10:500 TEA/HFIP/WFITM water or methanol, v/v/v.
hese appeared to be the strongest ion-pairing reagents readily available for testing, and
ppeared to be most functional for oligonucleotide analysis at the natural pH of ~8.
V. Analytical Column
The next most important element of chromatography for oligonucleotides is the
ate during elution is common practice
m
a
c
p
T
a
I
analytical column used for the assay. A low flow r
45
Figure 13: 25 mM imidazole/piperidine in MP, pH=8
There is limited initial retention of analyte, resulting in an analyte response in the solvent
front and lost response at the expected retention time.
46
Figure 14: 1:10:500 TEA/HFIP/MP, v/v/v, pH=4
There is no elution, as the pH is too strong for the oligonucleotide.
47
Figure 15: 1:10:500 TEA/HFIP/MP, v/v/v, pH=6
The pH is not yet basic enough for peak banding and elution strength
48
for oligonucleotide analysis, as it improves ionization of the compounds and allows f
more complete diffusion of the analyte through the TEA-laden sorbent bed of the
column.16 In addition, smaller pore size increases the selectivity of the column for oligos
of different size, and thus improves separation between compounds. However, column
used for this assay vary widely in the literature, and thus an investigation of the
performance of an array of columns was tested here.
The initial column tested was the Waters Xterra MS C18, 2.1 x 50 mm, 2.5 µm,
an industry favorite found in many of the literature articles detailing oligo analysis.3,16
However, the column tested here showed poor retention of the compound, resulting in
or
an
eak
his
,
.
polar component,
and the lowered response is expected. The Thermo-Scientific Betamax Base, 2.1 x 50
initial peak (height of 1.6 x 104 cps) of good shape, followed by a bleed of unbanded
compound from the column (Figure 16). While this column improves upon the ion-
pairing strength of standard C18 with a polymer mix of 2 silanol/1methyl group, the
sorbent is perhaps not uniform enough for the smooth elution of the oligonucleotide p
under these conditions, as silanol is not as strongly retentive for ion-pairing oligomer
analysis as the methyl groups. The next column tested, the Grace Division Discovery
Genesis C8, 2.1 x 50 mm, 3 µm, showed excellent retention, with a height of 4.4 x 106
cps and a retention time of 4.75 minutes (Figure 17). The strong performance of t
column is likely due to the monomeric, non-endcapped (and thus polar) silica sorbent
which is resistant to acid hydrolysis, highly selective, and stable under basic conditions
The Thermo-Scientific Hypersil Gold, 2.1 x 50 mm, 3 µm, column showed
similar response and good peak shape, but slightly less retention of the analyte band
(Figure 18); this column operates on pure silica interactions, without a
49
Figure 16: Waters Xterra MS C18, 50 x 2.1 mm, 2.5 µm
There is no retention of the analyte.
50
mm, 5 µm, column showed no banding ability whatsoever, resulting in a jumbled mess of
analyte response and no clear unique peak (Figure 19). As this column uses cyano groups
ound to the silica sorbent, showing affinity for amino groups and polar compounds
verall, a better performance was expected. However, upon further review, the methanol
oncentrations used during the chromatography of this assay may have been too high for
e proper function of this column. The Thermo-Scientific Aquasil C18, 2.1 x 50 mm, 3
m, column fared better with banding ability, but displayed incomplete elution strength
ith the gradient, resulting in a wide, jagged elution peak (Figure 20). Although the
ydrophilic endcapping of this column aids bonding with polar compounds, this sorbent
oes not appear to form strong enough bonds with the TEA of this ion-pairing assay. A
milar performance was noted from the Varian Pusuit Diphenyl, 2 x 50 mm, 5 µm,
isplayed poor, jagged peak shape in analyte elution (Figure 23). While the double bond
lectrons and 3 carbon (propyl) linker of this sorbent are somewhat successful, this
olumn is only presented for pH strength of 1.5-8, and thus not stable for this assay.
he Phenomenex columns as a group fared much better with selective, sharp peaks and
lean elution of the compounds from the columns. The Phenomenex Synergi Max-RP, 2
30 mm, 4 µm, column displayed a retention time of 3.9 minutes, and a peak height of
.5 x 106 cps (Figure 21). Even more impressive was the peak width of less than
0 seconds, indicating a clean elution of the compounds from the sorbent bed of the
olumn. However, this response is lower overall than the other Phenomenex columns,
kely due to the high hydrophobicity of this sorbent. The Phenomenex Synergi Polar-RP
performed almost identically to the Max-RP column, with a peak height of 2.5 x 106 cps,
b
o
c
th
µ
w
h
d
si
column, which showed good overall retention with a retention time of 4.4 minutes, but
d
e
c
T
c
x
2
3
c
li
51
Figure 17: Grace Division Discovery Genesis C8, 50 x 2.1 mm, 3 µm
There appears to be much improved retention, good peak shape and response.
52
Figure 18: Thermo-Scientific Hypersil Gold, 50 x 2.1 mm, 3 µm
The retention is not quite as good, with analyte break through at 1.8 minutes and lowered response resulting in principle peak
53
Figure 19: Thermo-Scientific Betamax Base, 50 x 2.1 mm, 5µm
This column results in poor banding and retention. Analyte continued to bleed from the olumn throughout elution. c
54
Figure 20: Thermo-Scientific Aquasil C18, 50 x 2.1 mm, 3 µm
This column produces incomplete elution at optimum gradients, resulting in slow,
consistent bleed of analyte from column and widened peak elution as well.
55
Figure 21: Phenomenex Synergi Max-RP, 30 x 2 mm, 4 µm
This column produces good retention and elution, although the response is not as high as Genesis column.
56
Figure 22: Phenomenex Synergi Polar-RP, 50 x 2 mm, 2 µm
The column displays a very sharp peak and good retention, although perhaps not as
retentive as Genesis due to its smaller surface area.
57
Figure 23: Varian Pursuit Diphenyl, 50 x 2 mm, 5 µm
T leading peak bl
his column produces poor banding and eed.
58
and a retention time of 4.4 minutes (Figure 22); nonetheless, this peak was wider,
indicating a larger area response of the compound. This column sorbent uses an ether-
linked phenyl group with polar endcapping, improving selectivity for polar and aromatic
com ance was seen in the Phenomenex Phenyl-hexyl,
2 x 50 mm, 3 µm, column, with a retention time of 4.8 minutes indicating strong sorbent
binding of the compound, and a peak height of 5.2 x 106 cps with a peak width of 15
seconds. This column truly out-performed the other columns in selectivity, strength of
binding, and overall peak shape (Figure 24). Phenyl-hexyl sorbents use the dense bonding
of the 6 carbon hexyl linker with no exposed silanol to increase retention of polar amine
ng the
ion-pai nown
in
e
pounds. However, the best perform
compounds, such as TEA, and improve separation; thus, these results mimic the expected
outcome.
V. Extraction Details
The testing of the reconstitution solution involved whether the compound would
be better able to bond to the sorbent when suspended in a solution already containi
ring reagents, or whether a pure water solution was more stable. Also unk
was whether the compound would bind to the plastic walls of a 96-well plate, with or
without ion-pairing reagent in the solution.
The results from this testing indicated that inclusion of the ion-pairing reagents
the reconstitution solution causes variability in the sample extracts from injection to
injection (Table 3). While the WFITM water samples showed a % Coefficient of Variation
of only 8.58 (ratio), the ion-pairing reconstitution samples showed a % Coefficient of
Variation of 65.6% by ratio. There appeared to be no improvement in response by using
this reconstitution solution for extracts, thus eliminating any advantag
59
Figure 24: Phenomenex Phenyl-hexyl, 50 x 2 mm, 3 µm
peak shape, with little to no bunching te res the bot
th
This guard produces good of analy ponse at tom of
e peak.
60
of the more capricious solution. Added ion pairing reagents in the reconstitution solution
kely increases binding of the compound to the walls of the storage units, through a
similar mechanism as the binding useful during LC loading, while being unnecessary
ple is fully inundated with ion-pairing reagents between the
utosampler and the column.
In addition, there appeared to be no improvement in sample consistency or
average response by the use of glass inserts for sample storage prior to injection. While
the % Coefficient of Variation was reasonable at 12.6%, the average response was much
lower than that of the samples stored in plastic wells prior to injection. Further testing of
these extracts by reinjecting on the days following the initial testing reproduced neither
the variability nor the previously witnessed average area responses, leading to the
conclusion that this storage condition is too inconsistent for use with sample extracts,
most likely due to analyte binding with the walls of the storage units.
VI. SPE Investigation
As previously tested SPE procedures had not yet yielded the sufficiently
optimized results for tissue and plasma analysis, a continued examination of possible SPE
testing was now initiated. Preliminary SPE testing indicated that the inclusion of ion-
pairing reagents was beneficial for higher average response and overall improvement of
tested samples. In addition, plasma extraction of any kind required a solid phase
extraction step for cleanliness. Extract components of plasma samples extracted solely by
liquid-liquid procedures clogged columns within 5-15 injections. To that end, a more
select group of ion-paired SPE procedures was tested.
The results from this testing indicated that while tissue extracts appeared to retain
li
overall, as the sam
a
61
Table 3: Comparison of Reconstitution Solutions and Storage Conditions
S mple Name Area IS Area Ratio_Area a IP Filtration Method SPE TEA Re -1 222 49983 0.635 con Plastic 1 135 3 IP Filtration Method SPE TEA Re 1-2 459 684con Plastic 1 920 330 2.133 IP Filtr TEA Re 1- 86 32ation Method SPE con Plastic 3 2 504 2194 0.889 Average 656186 452169 1.22 St Deviation 696 201537 798 0.80 % Coefficient of Variation 4106 4.6 65.6 IP Filtration Method SPE TEA Recon Gl 70 307073 ass 1-1 2 955 0.882 IP Filtration Method SPE TEA R s 1-2 0 94econ Glas 1 255 17 1.089 IP Filtration Method SPE TEA Recon Glass 1-3 13 12 799 176 1.133 Average 98337 109555 1.03 St Deviation 149503 171061 0.13 % Coefficient of Variation 156 12.6 152 IP Filtration Method SPE WFI Re c 1-1 217 256 con Plasti 261 745 0.846 IP Filtration Method SPE WFI Recon Plastic 1-2 206 229 369 128 0 901. IP Filtration Method SPE WFI Recon Plastic 1-3 228863 228703 1.001 Average 217497 238192 0.92 S 11249 16069 0.08 t Deviation % 5.17 6.75 8.58 Coefficient of Variation P iner less variable oveWFI recon less variable than IP re
lastic conta r time than glass container; con
62
sufficient matrix component material to stabilize SPE binding, the plasma extracts did not
able 4). The analyzed samples showed strong variability and poor (~10-20%) recovery,
while the load/wash “samples” displayed much higher response of the analyte. It was
clear that the m f the analyte was
and washing conditions, and thus was lost during the SPE procedure.
The analyte tested here is a much smaller oligonucleotide than has been
ly exam poor results of solid
hase extract othesizes smal n e backbo not h
nough bindi es to mak the io d S d. This re uch
rominent in sam hich p few al co nts for
analyte to use during sorbent binding (Table 5). However, the effect is still witnessed in
sue samples, which ppear to be sing up to roughly half of the analytes during
SPE loading an g. R and d response, due to extract cleanliness, in
e tissue samples are sufficient to offset these losse 6-7).
II. SPE Co parison
The IP filtration metho ot pr ansing benefits of a
standard SPE cedure, but fact i e ex t cleanliness ore s
ydro-Clean ry (binding analytes for washing prior to
lution), but sults in lower recovery due to insufficient binding of the test compound to
the sorbent bed. Recovery results were determined b ples
ith the resp samples s ith an ables 5- test r
dicate that extracts o in ap e t st rugged
ligonucleot Cyn us live bite over liquid-
(T
ajority o not sufficiently bound to the sorbent bed during
the loading
previous ined at PPD, Inc. The current theory concerning the
p ion hyp that the ler oligo ucleotid ne does ave
e ng sit e use of n-paire PE be sult is m mor e
p the plasma ples, w ossess er biologic mpone the
the tis a lo
d washin ecovery improve
th s (Tables
V m
d does n ovide as many cle
pro does in mprov trac . The m tandard
H method utilizes standard SPE theo
e re
y comparison of pre-spiked sam
w onse of piked w alyte in step 12 (T 7). The esults
in tissue nce aga pear to b he mo of the
o ide analysis. omolg r exhi d 71.0% rec y with liquid
63
Table 4: SPE R ts from Plasma Analysis
ple Name Area IS Area Ratio_Area
esul
Sam IP Filtration Method SPE Plasma 1-1 539110 2015323 0.268 IP Filtration Method SPE Pl a 1-2 971777 0.162 asm 157902 IP Filtration Me a 1-3 997904 0.217 thod SPE Plasm 216766 Average 304593 1328335 0.22 St Deviation 205219 595093 0.05 % Coefficient of 4 Variation 67. 44.8 24.6 Hydro-clean SPE Plasma 1-1 00 32090 83770 0.545 Hydro-clean SPE Plasma 1-2 29 73091 34293 0.421 Hydro-clean SPE Plasma 1-3 28 1634933 0.468 7646Average 427586 917665 0.48 St Deviation 49 62961 45424 0.06 % Coefficient n of Variatio 69.3 70.3 13.1 Load/Wash Fr ation E Plas 756026 action IP Filtr M Pethod S ma 1-1 822961 0.919 Load/Wash Fr n M PE Plasaction IP Filtratio ethod S ma 1-2 1076445 1559199 0.69 Load/Wash Fr n M PE Plasmaction IP Filtratio ethod S a 1-3 933872 1196691 0.78 Average 922114 1192950 0.80 St Deviation 3 16053 368133 0.12 % Coefficient of Variation 17.4 30.9 14.5 Load/Wash Fraction Hydro-clean SPE Plasma 1-1 222079 297084 0.748 Load/Wash Fraction Hydro-clean SPE Plasma 1-2 510864 458179 1.115 Load/Wash Fraction Hydro-clean SPE Plasma 1-3 484780 490853 0.988 Average 405908 415372 0.95 St Deviation 159733 103735 0.19 % Coefficient of Variation 39.4 25.0 19.6 Plasma SPE highly variable and most of analyte is lost in the loading and washing steps
64
Table 5: Plasma Recovery with SPE Methods
Hydro-C
Sample ID
ARe
Int. StRespon
mple ID ost-action ified)
e lean SPE
(pre-extraction fortified)
nalyte sponse
d. se
Sa(p
extrfort
AnalyteRespons
Int. Std. Response
Plasma
PRE 1 2823117 3443051 POST Pre-Evap 1-1 435 7625545 1- 8176
PRE 1-3 2551735 37ST Pre-
3 8990137 8342773 54354 Evap 1-PO
Mean 266419 489 3 7887 Mean 8766786 7912472 SD 14152 4 37 9 2 7119 SD 516279 9501
%
Var ion 5.31% 7.09%
Coefficient of Variation 5.89% 4.80%
Coefficientof
iat
%
very 30.4% 44.1% %Reco
Hydro-Clean thod results in ~30-40% recovery, but has
ess
me
higher response due to extract cleanlin
LLE SPE Filtrati
Sample ID (pre-
extraction fortified)
Analyte Response
Int. SRespo
Sample ID (post-
n Analyte Response
Int. Std. Response on
td. nse
extractiofortified)
Plasma
PRE 1-1 2739838 1956T Pre- 1-1 11 040
POSEvap 79532 7066638
PRE 1-2 3882580 2470985 POST Pre-Evap 1-2 3550907 2960394
PRE 3 2206157 1230426 POST Pre-Evap 1-3 903 3969364 1- 5078
Mean 2942858 188 5527674 4665465 5817 Mean SD 8564 2 21353 6 3254 SD 2235199 9796
% Coof Var
fficient
iation 40.
efficient
iation 29.1% 33.0% of Var
% Coe
4% 45.9% Recovery % 53.2% 40.4%
2 19 0 POST Post-Evap 1-1 125 PRE 1-1 739838 5604 5350 5706433
P 38 24ost-2 730 RE 1-2 82580 70985
POST PEvap 1- 6829 7077423
PRE 3 22061 7 12 Post-
1-3 853 1- 5 30426 POSEvap
T5743 6405980
Mean 2942858 1885817 Mean 5974569 6396612 SD 856453 623254 SD 766309 685543 LLE Filtration Method has 40-54% recovery prior to evaporation, and 30-50% recovery post-evaporation.
% Coefficient of Variation 29.1% 33.1%
% Coefficient of Variation 12.8% 10.7%
Testing conducted without ethylene glycol %Recovery 49.3% 29.5%
65
Table 6: Liver Recovery
(pre-extraction Analyte Int. Std.
(post-extraction Analyte Int. Std.
Sample ID
fortified) Response Response
Sample ID
fortified) Response
Response LLE 0.22 µm
filter Liver
PRE 1-1 944699 955454 Evap 1-1 1636398 1702855 POST Pre-
PRE 1-2 1129028 1630865 POST Pre-Evap 1-2 1462981 1761765
PRE 1-3 1422060 1612087 POST Pre-Evap 1-3 1827360 2173228
Average 1516435 1840515 0.826 St Deviation 62820 127905 0.068 % Coefficient of Variation 4.14 6.95 8.19
Having IP Filtration reagents in the sales and elution solution appears beneficial for response and consisten y
ction of Io ring Reage rior to g and Elutio
Sample Nam rea
Liver No TEAA Addition Pre 1-2 584 Liver No TEAA Addition Pre 1-3 .561 Liver No TEAA Addition Pre 1-4 .599 Averag 148960 0.581 St Dev 7019 7170 0.019 % Coe Variation 4.7 2.8 3.29 Liver P 1641Liver Pre 1-2 0.818 Liver Pre 1-3 159943 0.783 Liver Pre 1-4 0.81
St Dev 6598 0.018 % Coe 4.0 2.6 2.28 Liver No IP Filtr on Elution Pr 1-1 0.72 Liver No IP Filtration Elution Pr 1-2 152707 0.769 Liver No IP Filtration Elution Pr 1-3 144897 0.809 Liver No IP Filtration Elution Pr 1-4 157325 0.901
c
71
Table 10: Ethylene Glycol Recovery Table
mple ID (pre-
extraforti
Analytespon
t. Std. ponse
D (post-
ction fied)
lyte nse
. e
Sa
ction fied) R
e se
InRes
Sample I
extraforti
AnaRespo
Int. StdRespons
LLE SPE filtration
Plasma PRE No EtGly 1-1 2295520 1527
POST EtGly vap 1-1 5641456 4273498 645 Pre-E
PRE No EtGly 1-2 1554035 1107
T EtGly vap 1-2 5580104 4238137 598 Pre-E
POS
PRE No EtGly 1-3 788974 613
T EtGly vap 1-3 6149238 4794177 094 Pre-E
POS
Mean 1546176 1082779 Mean 5790266 4435271
% oefficient
of Variation 48.7% 42.3%
ficient ation 0%
C
of Vari% Coef
5.4 7.02% %Recovery 26.7% 24.4%
PRE EtG1-1 5244013 3969
T EtGly ap 1-1 5641456 4273498
ly 741 Pre-Ev
POS
PRE EtG1-2 5255362 3531690
T EtGly ap 1-2 5580104 4238137
ly POSPre-Ev
PRE EtG1-3 5200839 3029175
T EtGly Pre-Evap 1-3 6149238 4794177
ly POS
Mean 5233405 3510202 Mean 5790266 4435271
% Coefficienof Variation 0.550% 13.4%
% Coefficient of Variation 5.40% 7.02%
t
%Recovery 90.4% 79.1% LLE Filtration Method has 80-90% recovery and low variability with ethylene glycol added to sample during evaporation
72
Table 11: Ethylene Glycol in Tissue Extraction
Area IS Ar tio_Area Sample Name ea RaIP FILTRATION Kidney Pre 1-1 1232 0685 1.184 453 104IP FILTRATION Kidney Pre 1-2 882 6731 1.214 351 72IP FILTRATION Kidney Pre 1-3 1004 9097 1.093 510 91IP FILTRATION Kidney Pre 1-4 926 8093 1.032 830 89IP FILTRATION Kidney Pre 1-5 1279 4612 1.191 777 107IP FILTRATION Kidney Pre 1-6 9099 7309 0.941 48 96Average 1039312 7754 1.109 93St Deviation 173414 123815 0.108 % Coefficient of Variation 1 13.2 9.69 6.7 IP FILTRATION EtGly Kidney Pre 1-1 479 8851 0.706 567 67IP FILTRATION EtGly Kidney Pre 1-2 680 1239 0.666 654 102IP FILTRATION EtGly Kidney Pre 1-3 924 3610 0.837 249 110IP FILTRATION EtGly Kidney Pre 1-4 94027 5253 0.807 4 116IP FILTRATION EtGly Kidney Pre 1-5 955005 4400 0.8 119IP FILTRATION EtGly Kidney Pre 1-6 762370 1091400 0.699 Average 790353 1042459 0.753 St Deviation 188106 188128 0.071 % Coefficient of Variation 2 18.1 9.37 3.8 IP FILTRATION Kidney Post 1-1 2103 6667 1.092 173 192IP FILTRATION Kidney Post 1-2 1649 0779 0.877 716 188IP FILTRATION Kidney Post 1-3 1647 7574 0.965 032 170IP FILTRATION Kidney Post 1-4 200188 0405 1.042 5 192IP FILTRATION Kidney Post 1-5 1938733 3122 0.968 200IP FILTRATION Kidney Post 1-6 1923392 1854213 1.037 Average 1877 2127 0.997 322 188St Deviation 188 9337 0.076 285 9% Coefficient of Variation 1 5.28 7.63 0.0 IP FILTRATION EtGly Kidney Post 1-1 1816 7028 1.005 456 180IP FILTRATION EtGly Kidney Post 1-2 21310 6087 1.101 57 193IP FILTRATION EtGly Kidney Post 1-3 1860882 4734 1.037 179IP FILTRATION EtGly Kidney Post 1-4 1223423 1348732 0.907 IP FILTRATION EtGly Kidney Post 1-5 2187722 1960204 1.116 IP FILTRATION EtGly Kidney Post 1-6 2271586 1935510 1.174 Average 1915187 1797049 1.057 St Deviation 384481 230745 0.095 % Coefficient of Variation 20.1 12.8 8.95 Ethylene glycol addition not advisable in tissue extractions
-72. . Gilar, M.; et al. Oligonucleotides. 2003, 13, 229-243.
.davidson.edu/Courses/Molbio/MolStudents/spring2003/Holmbergligonucleotide_synthesis.htm. 16 Jun 2008.
. T/2000/Septem l. 16 Jun 2008. 7. holas M, and B gene. 2003, 22, 9087-9096. 8. Y.; et al. Anal. Che , 6023-6028. 9. moczko JL St Cell Biology
3. Fountain, K.; et al J. Chromatogr. B. 2003, 783, 6145. http://www.bio/o6 http://www.abrf.org/JB
an, Nicber00/sep00bintzler.htm
ennett, C Frank. Onco De Yu, m. 2003, 75 (21)
ryer L. M Berg JM, Ty olecular , 5th ed. New York: 2002. 10 vid. Mass spect desk reference. Sparkman, O. Da rometry . 1st ed. Pittsburgh: 2000. 11 ar, M.; Gebler, Commun. Mass Spectrom. 2004, 18, 1212 Pure & Appl. Chem No. 4, 819-872. 13 ouglas T. Ion Chromatography
. Fountain, K.; Gil J. Rapid95-1302. . Ettre, L.S. . Fritz, Jame
.1993, 65, s S, and Gjerde, D 3rd ed. New
Y14 ractical Hi ance Liquid Chromatography.
ork:2000. . Meyer, Veronika R. P gh-Perform 2nd ed.
Chichester, ENG: 1994. 15 ; et al. J. Chr 27. 16 t al. J. Chromatogr. A. 8, 167-182. 7 . Analytical Biochemistry. 8, 196-206.
cCloskey, J. Current Opinion in Biotechnology. 1998, 9, 25-34. .; Gilar, M.; Gebler, J. Rapid Commun. Mass Spectrom. 2003, 17,
6420. 1996, 7,21. Gilar, M.; Belenky, A.; W 2001, 921, 3-13. 22 lcher, W J. Mass Spectrom. 2003, 38, 108-116. 23 Lin, J ivasan, Karthik. Anal. Chem. 2007, 79, 3416-34
. Esmans, E.
. Gilar, M.; eomatogr. A 8, 794, 109-1. 199
2002, . Gilar, M
95 2001, 291
18. Crain, P.; M19. Fountain, K
6-653. Muddiman, D.; Cheng, X.; Udseth, H.; Sm
697-706. ith, R. J Am Soc Mass Spectrom.
ang, B. J. Chromatogr. A.. Oberacher, H.; Wa. Zhang, Guodong;
.; Huber, C. ian; Srin
24.
96
APPENDIX A: Original Method
P T e to be thawed n ice through Step 3. Final sample ex stored at 2 to 8
•For individual tissue samples, mince a 50-100-mg portion of sample tissue, mg into a Fast d add 80 µL of homogenization buffer. Add
en bea the tube and mix .
d Q fer a 100-µL aliquot of blank matrix bulk homogenate to a FastPrep tube containing ~¼” of matrix beads. Add 20 µL of the
control working solution into the a iate tu
•For matrix bla ansf aliqu atrix bulk homogenate to a stPrep tube aining ~¼” trix beads. Add 20 µL of W I water into the ropriate tub
Plasma samples are to be d on ice. mple ex cts are to be at 2 to 8 °C.
• Fo ndividual p sample ot a 10 sample to a Phase Lock gel tube. Add 20 µL of WFI water. A L internal standard working solution to all samples. Proceed to step 4.
sfer a 100-µL aliquot of blank matrix to a Phase Lock gel tube. Add 20 µL of the appropriate calibrati quality c work n priate tu /mL internal standard working s sample ed to step
• Fo atrix blan nsfer a 1 liquot of blank matrix to a Phase Lock gel e. Add 20 µ FI wate e appr ate tube. Add L of 2000 ng/ internal sta working n to all ples except blanks without internal standard. Add 20 µL of W ter to ks without internal standard. Proceed to step 4.
rine samp are to be t on ice. F ple extracts are to be sto at 2 to 8 °C.
• For individual urine samples, aliquot a 100-µL sample to a Phase Lock gel tube. Add 20 µL of WFI water. Add 20 µL of 2000 ng/mL internal standard working solution to all samples. Proceed to step 4.
• For calibrators and QCs, transfer a 100-µL aliquot of blank matrix to a Phase Lock gel tube. Add 20 µL of the appropriate calibration standard or quality
rocedure
issue samples artracts are to be
and processed o °C.
weigh 20 Prep tube, an~¼” of matrix grevigorously briefly
ds. Add 20 µL of WFI water. Cap
•For calibrators an Cs, trans
appropriate calibration standard or quality ppropr be.
nks, tr er a 100-µL ot of blank m Fa cont of ma Fapp e.
thawe Final sa tra stored
r i lasma s, aliqu 0-µLdd 20 µL of 2000 ng/m
• For calibrators and QCs, tranon standard or
20 µL of 2ontrol ing n i solutio to the approlution
o be. Addroce
000 ng to all s. P 4.
r m ks, tra 00-µL atub L of W r into th opri 20 µmL ndard solutio sam
FI wa blan
U
les hawed inal sam red
97
control working solution into the appropriate tube. Add 20 µL of 2000 ng/mL internal stand so samples. Proceed to step 4.
• Fo matrix bl ansfe aliquot of blank matrix to a Phase Lock gel e. Add 20 µ FI wate e app ate tube. Add 2 L of 2000 ng/m internal stan working to all ples except blanks without internal standard. Add 20 µL of W ter to b s without internal standard. Proceed to step 4.
rocedure S
. Add 20 µL of 2000 ng/mL internal standard working solution to all samples except blank ta dd 20 ater to blanks without internal standard. M rously brie .
2. Cap and hom ize in the Fa rep app ed of 5.5 for two cycles of 30
s. Monitor heat build-up of the samples apparatus and cool sample
. Transfer each homogenate sample to a Phase Lock gel tube. 4. Add 200 µL of WFITM water to eac 5. Add 100 µL of concentrated ammo %, VWR Scientific) to
each sample
5:24:1 phenol/chloroform/isoamyl alcohol (Fluka) to each sample. be several times
7. min. 8. chloroform (Sigma) to each sample. 9. be several times igorously for 30 s, and then centrifuge at
r 5 min. 10 00 rpm for 5 min. 11 t to an Ul C filtration tubes, 0.22 µm, and
2 min. 12 e appr e well of a 96-position, 2.0-mL, square-
n . 13 nder a nitrogen stream at app ately 45 °C.
ard working lution to all
r anks, tr r a 100-µLtub L of W r into th ropri 0 µ
L dard solution samFI wa lank
P teps
1s without internal s ndard. A µL of WFI w
ix vigo fly
ogen stP ara spe in the FastPrep
tus at a
tubes on ice as necessary. 3
h sample.
nium hydroxide (28-30.
6. Add 300 µL of 2
Gently rock each tu and then mix vigorously for 30 s.
Centrifuge at 15000 rpm for 5
Add 300 µL of
Gently rock each tu , mix v15000 rpm fo
. Centrifuge at 150
. Transfer the aqueous extrac trafree-Mcentrifuge at 15000 rpm for
. Transfer the aqueous extract to th opriatwell, conical-bottom, polypropyle e plate
. Evaporate u roxim
98
14. Reconstitute with 100 µ d mix vigorously for approximately 30 s.
15. Inject 10 µL of extract on the mass spectrometer for each sample.
ptimal ranges may vary for each LC/MS system. Mass s Autosa
L of WFITM water. Seal the plate an
Instrument Parameters O
pectrometer: Sciex 4000, using Analyst software.
mpler Method
AutoCyclSyrinSampLC MVoluNeedle Stroke:
1000 µL 35 µL/s
Sampling Syringe Speed: 15 µL/s
RR 5.0 s Purge Time: 5.0 min
hromatography
sampler: Shimadzu SIL-HTC e: LC-Inj ge Standard Loop Volume: 10 µL ixing Chamber
me: 10 µL
50 mm Rinse Volume: Rinsing Syringe Speed:
Cooler Temperature: 2 to 8 °C inse Mode: Before and After Aspiration inse Diptime:
C
A enex Phenyl-hexyl 50 x 2 mm, 3 µm, Product No. 00B-4256-B0
C ture: 60 °C
t
Mobile Phase A, Load 0.5:5.0:500 TEA/HFIP/WFI Water, v/v/v, with 10 µm
Composition Step Total Time Flow Rate (min) (µL/min) A (%) B (%) 0 0.00 0.200 100 0 1 3.00 0.200 75 25 2 6.50 0.200 66 34 3 7.50 0.200 100 0 4 11.00 0.200 100 0
Valco Valve A Program (Loading/Eluting/Washing)
Total Time (min) Position CommentsInitial B Load Sample 2.00 A Elute 6.20 B Wash Column
Valco Valve B Program (Divert/Make-Up)
min) Position Comments
Total Time (Initial B Make-Up Flow to MS3.50 A Elute Sample to MS6.20 B Make-Up Flow to MS
Mass Spectrometry
ass Spectrometer: M Sciex API 4000, Triple quadrupole LC/MS/MS Ionization Mode: Electrospray 4, MRM, negative ion
RResolution Q3: Unit Ion Energy 1 (1E1) -2.00
Quantitation: Based on peak area Calibration: PPGs
: 500 °C IonSpray Voltage: -4500 V
9.00 Curtain Gas Flow (CUR): 30.0 NebuTurbDeflPausAcqu
CAD, CUR, NEB, AUX Gas: Nitrogen esolution Q1: Unit
Ion Energy 3 (1E3) -2.20
Ion Source Temp
Electron Multiplier (CEM): 2400 V Collision Gas Flow (CAD):
lizer Gas Flow (NEB/GS1): 40.0 o IonSpray Gas (AUX/GS2): 50.0 ector Potential (DF): 100 e Time: 5 ms isition Time: 11.0 min
101
APPENDIX B Optimized Method
Tis ocessed on ice through Step 3. Final sample xtracts are to be stored at 2 to 8 °C.
•For individual tissue samples, mince a 50-100-mg portion of sample tissue, d
he tube and mix vigorously briefly.
fer a 200-µL aliquot of blank matrix bulk g ~¼” of matrix beads. Add 20 µL of the y control working solution into the
s, transfer a 200-µL aliquot of blank matrix bulk homogenate to a FastPrep tube containing ~¼” of matrix beads. Add 20 µL of WFI water into
rocedure Steps
. tus at a speed of 5.5 for two cycles of 30 ratus and cool sample
h
l samples except ks without internal
sly briefly.
ix vigorously for 1 min in the Phase
centrated ammonium hydroxide to each sample.
7. f 25:24:1 phen l alcohol to each sample. Gently ach tube several times and x vigorously for 30 s.
8. 5000 rpm for 2 min.
Tissue Procedure
sue samples are to be thawed and pre
weigh 20 mg into a FastPrep tube, and add 480 µL of homogenization buffer. Ad~¼” of matrix green beads. Add 20 µL of WFI water. Cap t
•For calibrators and QCs, transhomogenate to a FastPrep tube containinappropriate calibration standard or qualitappropriate tube. Add 300 µL of homogenization buffer to each tube.
•For matrix blank
the appropriate tube. Add 300 µL of homogenization buffer to each tube.
P
Cap and homogenize in the FastPrep appara1s. Monitor heat build-up of the samples in the FastPrep appatubes on ice as necessary.
2. Transfer a 200-µL aliquot of each omogenate sample to a Phase Lock gel tube. 3. Add 20 µL of 2000 ng/mL internal standard working solution to al
blanks without internal standard. Add 20 µL of WFI water to blanstandard. Mix vigorou
4. Add 200 µL of chloroform to each sample, and m
Lock gel tube. 5. Centrifuge at 15000 rpm for 2 min. 6. Add 100 µL of con
Add 300 µL o ol/chloroform/isoamyrock e then mi
Centrifuge at 1
102
9. µL of chloroform to each sample. 10 ch tube several time trifuge at
11 rpm for 2 min 12 ueous extract to an Ultrafree-MC filtration tubes, 0.22 µm, and
t 15000 rpm for 2 min.
ueous extract to the appropriate well of a 96-position, 2.0-mL, square-cal-bottom, polypr
14 r a nitrogen 15. Reconstitute with 200 µL of WFI water. Seal the plate and mix vigorously for
. 16. Inject 10 µL of extract on the mass spectrometer for each sample.
P Plasma samples are to be thawe ocessed on ice through Step 1. Final sample ex red at 2 to 8
tissue el tube. Add 20 µL of WFI
nd Q sfer a 200-µL aliquot of blank matrix to a Phase gel tube. Add 20 µL of the appropriate calibration standard or quality
lution propriate tube.
atrix blanks, transfer a 200 liquot of blank matrix to a Phase Lock e. Add 20 µL of W he appropriate tube.
1. 0 ng/mL i tandard working solution to all samples except
of WFI water to blanks without internal ix vigorously b
2. each sample. . Add 100 µL of concentrated ammonium hydroxide to each sample.
Add 200
. Gently rock ea s, mix vigorously for 30 s, and then cen15000 rpm for 5 min.
. Centrifuge at 15000
. Transfer the aqcentrifuge a
13. Transfer the aq
well, coni opylene plate.
. Evaporate unde stream at approximately 45 °C.
TM
approximately 30 s
lasma Procedure
d and prtracts are to be sto °C.
• For individual samples, aliquot a 200-µL sample to a Phase Lock g water.
• For calibrators aLock
Cs, tran
control working so
• For
into the ap
mgel tub
-µL aFI water into t
Add 20 µL of 200 nternal sblanks without internal standard. Astandard. M
dd 20 µLriefly.
Add 200 µL of homogenate buffer to
3
103
4. Add 400 µL of 25:24:1 phenol/chloroform/isoamyl alcohol to each sample. Gently rock each tube se n igorously for
5. Centrif at 15000 r 2 6. Add 40 L of chloro o each sa 7. Gently rock each tube several times, orously r 30 s, and the trifuge at
15000 rpm for 5 min. 8. Centrifuge at 15000 rpm for 2 min . Add 100 µL of 1 M TEAA (Fluka) to each sample.
10. Trans aque o iate SPE filtra g the load step.
11. SPE extraction:
i. IP F on Metho dition LB 10 mg cartridge with 1.0 mL MeOH, and then 1.0 mL 8 m EA/100 mM P in water. App dium vac etween ditioning washes. Load sam nto the ap te well of a 30 mg HLB SPE plate CA OAD A UTION VOLUMES FORBL OWN. Lo sample wly. “Elute” w 1.0 mL
TEA, v/v. Apply low vacuum for elution.
12. Add 10 µL of ethylene glycol to ate under a nitrogen stream at app ately
13. Reconstitute with 200 µL of WFI water. Transfer extracts to a plastic well in a 96-
well plate. Seal the plate and mix vigorously for 30 sec, then centrifuge at 3500 for 2 minutes.
14. Inject 10 µL of extract on the mass spectrometer for each sample.
Urine Procedure Urine samples are to be thawed and processed on ice through Step 1. Final sample extracts are to be stored at 2 to 8 °C.
• For individual tissue samples, aliquot a 200 µL sample to a Phase Lock gel tube. Add 20 µL of WFI water.
• For calibrators and QCs, transfer a 200-µL aliquot of blank matrix to a Phase Lock gel tube. Add 20 µL of the appropriate calibration standard or quality control working solution into the appropriate tube.
veral times a d then mix v 30 s.
uge rpm fo min.
0 µ form t mple.
mix vig fo n cen
9
fer the ous extract t the appropr tion tube durin
iltrati d: Con an HM T con
HFIly me uum bples i propriaTCH L ND EL OWD ad the slo ith
70:30 MeOH/8 mM
each sample and evaporro imx 45 °C.
104
• For matrix blanks, transfer a 200-µL aliquot of blank matrix to a Phase Lock gel tube. Add 20 µL of WFI water into the appropriate tube.
Procedure Steps 1. Add 20 µL of 2000 ng/mL internal standard working solution to all samples except
blanks without internal standard. Add 20 µL of WFI water to blanks without internal standard. Mix vigorously briefly.
2. Add 100 µL of concentrated ammonium hydroxide to each sample. 3. Add 400 µL of 25:24:1 phenol/chloroform/isoamyl alcohol to each sample. Gently
rock each tube several times and then mix vigorously for 30 s. 4. Centrifuge at 15000 rpm for 2 min. 5. Add 400 µL of chloroform to each sample. 6. Gently rock each tube several times, mix vigorously for 30 s, and then centrifuge at
15000 rpm for 5 min. 7. Transfer the aqueous extract to an Ultrafree-MC filtration tubes, 0.22 µm, and
centrifuge at 15000 rpm for 2 min. 8. Transfer the aqueous extract to the appropriate well of a 96-position, 2.0-mL, square-
well, conical-bottom, polypropylene plate. 9. Evaporate under a nitrogen stream at approximately 45 °C. 10. Reconstitute with 200 µL of WFITM water. Seal the plate and mix vigorously for
approximately 30 s. 11. Inject 10 µL of extract on the mass spectrometer for each sample.
Instrument Parameters Optimal ranges may vary for each LC/MS system. Mass spectrometer: Sciex 4000, using Analyst software. Autosampler Method
Needle Stroke: 50 mm Rinse Volume: 1000 µL Rinsing Syringe Speed: 35 µL/s Sampling Syringe Speed: 15 µL/s Cooler Temperature: 2 to 8 °C Rinse Mode: Before and After Aspiration Rinse Diptime: 5.0 s Purge Time: 5.0 min
Chromatography
LC Pump: HP 1100 Series or Shimadzu LC-10AD VP
Guard Column: Phenomenex Phenyl-hexyl 4.0 x 3.0 mm, Product No. AJO-4351.
Analytical Column: Phenomenex Phenyl-hexyl 50 x 2 mm, 3 µm, Product No. 00B-4256-B0
Column Temperature: 60 °C
Pump Program: Gradient
Mobile Phase A, Load Mobile Phase:
1.0:10:500 TEA/HFIP/WFI Water, v/v/v, with 10 µm
EDTA
Mobile Phase B: Methanol
Wash Mobile Phase: 0.5:5.0:150:350 TEA/HFIP/WFI Water/Methanol,
Total Time (min) Position Comments Initial B Load Sample 2.00 A Elute 6.20 B Wash Column
108
Valco Valve B Program (Divert/Make-Up)
Total Time (min) Position Comments Initial B Make-Up Flow to MS3.50 A Elute Sample to MS6.20 B Make-Up Flow to MS
Mass Spectrometry
Mass Spectrometer: Sciex API 4000, Triple quadrupole LC/MS/MS Ionization Mode: Electrospray 4, MRM, negative ion CAD, CUR, NEB, AUX Gas: Nitrogen Resolution Q1: Unit Resolution Q3: Unit Ion Energy 1 (1E1) -2.00 Ion Energy 3 (1E3) -2.20 Quantitation: Based on peak area Calibration: PPGs Ion Source Temp: 500 °C IonSpray Voltage: -4500 V Electron Multiplier (CEM): 2400 V Collision Gas Flow (CAD): 9.00 Curtain Gas Flow (CUR): 30.0 Nebulizer Gas Flow (NEB/GS1): 40.0 Turbo IonSpray Gas (AUX/GS2): 50.0 Deflector Potential (DF): 100 Pause Time: 5 ms Acquisition Time: 11.0 min