an evaluation of extraction parameters and lcms analysis...

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

Acknowledgements..............................................................................................................v

List of Tables ..................................................................................................................... vi

List of Figures ................................................................................................................... vii

Introduction..........................................................................................................................9

I. Oligonucleotide Overview....................................................................................9

II. History of Oligonucleotide Analysis.................................................................13

III. Chromatographic Considerations.....................................................................14

IV. Guidelines for Intended Assay Improvement ..................................................16

Methodology......................................................................................................................18

Experimentation.................................................................................................................20

I. Detergent Determination ....................................................................................20

II. Initial SPE Examination ....................................................................................21

III. Mobile Phase Components ..............................................................................23

IV. Analytical Column...........................................................................................23

V. Extraction Details..............................................................................................24

VI. SPE Investigation.............................................................................................24

VII. SPE Comparison.............................................................................................25

VIII. Urine Analysis...............................................................................................27

IX.Guard Column Analysis ...................................................................................27

X. Tissue Specifics.................................................................................................28

Results and Discussion ......................................................................................................28

ii

I. Detergent Determination ....................................................................................28

II. Initial SPE Examination ....................................................................................30

III. Mobile Phase Components ..............................................................................38

IV. Analytical Column...........................................................................................45

V. Extraction Details..............................................................................................59

VI. SPE Investigation.............................................................................................61

VII. SPE Comparison.............................................................................................63

VIII. Urine Analysis...............................................................................................70

IX.Guard Column Analysis ...................................................................................77

X. Tissue Specifics.................................................................................................86

Conclusions........................................................................................................................94

Literature Cited ..................................................................................................................96

Appendices................................................................................................................. 97-108

A Original Method.................................................................................................97

B Optimized Method............................................................................................102

iii

Abstract This thesis represents a comprehensive investigation of the extraction and

instrumentation parameters of oligonucleotide analysis, using the various chemical

properties associated with these nucleic acids. Various research groups have performed

much background analysis, and this information will be summarized in the introduction.

This research represents years of scientific experimentation, and yields a number of

accepted practices in the study of oligonucleotides. From this starting base of knowledge,

further advancements and improvements to the analysis were pursued.

The initial area of examination was the extraction procedure. Key areas of the process

were improved, from tissue preparation to the optimization of a solid-phase extraction

procedure. In tandem with the extraction optimization, the instrumentation of the MS/MS

analysis was investigated. Column optimization, mobile phase selection, and effective

maintenance of chromatography were examined, while utilizing the scientific techniques

of separation science and chromatographic ion-pair binding to guide the assay

parameters. Once these areas were streamlined, multiple matrices were analyzed to

evaluate the overall success of the new technique in the pivotal task of overcoming

different matrix effects. Each matrix required a unique variation of the overall method in

order to achieve optimum results, with urine being the simplest matrix extraction and

plasma the most complex. Tissue matrix created a distinctive challenge, with different

organ types exhibiting variable extraction characteristics. However, acceptable extraction

and instrument parameters were developed for all tested matrices without exception.

iv

Acknowledgements

The author would like to thank her wonderful husband, Paul, and loving family.

She would also like to gratefully acknowledge the Department of Chemistry and

Biochemistry at the University of North Carolina-Wilmington and her employers at PPD,

Inc, for the opportunity to fulfill the requirements of this wonderful program. She would

like to thank in particular Dr. Bruce Petersen at PPD, Inc, and Dr. John Tyrell and Dr.

James Reeves at University of North Carolina-Wilmington, for their generous guidance

and support throughout the program.

v

List of Tables Table Page

Table 1: Buffer Detergent Data..........................................................................................35

Table 2: Initial SPE Comparison. ......................................................................................37

Table 3: Comparison of Reconstitution Solutions and Storage Conditions. .....................62

Table 4: SPE Results from Plasma Analysis. ....................................................................64

Table 5: Plasma Recovery with SPE methods. ..................................................................65

Table 6: Liver Recovery. ............................................................................................. 66-67

Table 7: Kidney Recovery. ................................................................................................68

Table 8: Addition of TEAA to LLE Filtration...................................................................71

Table 9: Introduction of Ion-pairing Reagents Prior to Loading and During Elution. ......71

Table 10: Ethylene Glycol Recovery Table.......................................................................72

Table 11: Ethylene Glycol in Tissue Extraction. ...............................................................73

Table 12: Plasma Run. .......................................................................................................74

Table 13: Urine Recovery..................................................................................................75

Table 14: Urine Data..........................................................................................................76

Table 15: Urine Run...........................................................................................................78

Table 16: Tissue Comparison Between a Kidney Calibration Curve and Kidney and Liver

QCs. ...................................................................................................................................87

Table 17: Tissue Comparison (Low Level). ................................................................ 89-90

Table 18: Tissue Comparison (High Level)................................................................. 91-92

Table 19: 40% Matrix Portioning Extraction.....................................................................93

vi

List of Figures Figure Page

Figure 1: Nucleic Acid Pictorial ........................................................................................10

Figure 2: Oligonucleotide Synthesis..................................................................................11

Figure 3: Antisense Oligonucleotides in Action................................................................12

Figure 4: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% CHAPS ..............................31

Figure 5: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% NP-40 Alternative..............32

Figure 6: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% SDS....................................33

Figure 7: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% Tween-80...........................34

Figure 8: 0.5:5.0:500 TEA/HFIP/MP, v/v/v. .....................................................................40

Figure 9: 1:10:500 TEA/HFIP/MP, v/v/v ..........................................................................41

Figure 10: 1:10:500 TBA(tributylamine)/HFIP/MP, v/v/v................................................42

Figure 11: 1:10:500 TPA(tripropylamine)/HFIP/MP, v/v/v..............................................43

Figure 12: 1:10:500 DEA(diethylamine)/HFIP/MP, v/v/v ................................................44

Figure 13: 25 mM imidazole/piperidine in MP, pH=8 ......................................................46

Figure 14: 1:10:500 TEA/HFIP/MP, v/v/v, pH=4.............................................................47

Figure 15: 1:10:500 TEA/HFIP/MP, v/v/v, pH=6.............................................................48

Figure 16: Waters Xterra MS C18, 50 x 2.1 mm, 2.5 µm .................................................50

Figure 17: Grace Division Discovery Genesis C8, 50 x 2.1 mm, 3 µm ............................52

Figure 18: Thermo-Scientific Hypersil Gold, 50 x 2.1 mm, 3 µm ....................................53

Figure 19: Thermo-Scientific Betamax Basic, 50 x 2.1 mm, 5µm....................................54

Figure 20: Thermo-Scientific Aquasil C18, 50 x 2.1 mm, 3 µm.......................................55

Figure 21: Phenomenex Synergi Max-RP, 30 x 2 mm, 4 µm............................................56

Figure 22: Phenomenex Synergi Polar-RP, 50 x 2 mm, 2 µm...........................................57

vii

Figure 23: Varian Pursuit Diphenyl, 50 x 2 mm, 5 µm .....................................................58

Figure 24: Phenomenex Phenyl-hexyl, 50 x 2 mm, 3 µm .................................................60

Figure 25: C18 Guard, 4 x 2 mm .......................................................................................80

Figure 26: C18 Guard, 4 x 3 mm .......................................................................................81

Figure 27: Gemini C18Guard, 4 x 2 mm ...........................................................................82

Figure 28: Polar RP Guard, 4 x 3 mm ...............................................................................83

Figure 29: Phenyl-hexyl Guard, 4 x 2 mm ........................................................................84

Figure 30: Phenyl-hexyl Guard, 4 x 3 mm ........................................................................85

viii

Introduction

I. Oligonucleotide Overview

As science improved the synthesis of chemically modified oligonucleotides, the

bioanalytical world of oligonucleotides widened sufficiently, requiring new quantitation

techniques. Oligonucleotides, also termed nucleic acids, are linear sequences of

nucleotides, which consist of a heterocyclic nitrogenous base, a sugar and a phosphate

group.1 There are five bases utilized in nucleic acids (DNA or RNA): cytosine (C),

thymine (T), and guanine (G), which are pyrimidine bases, and adenine (A) and uracil

(U), which are purine bases. DNA consists of ACGT, and uses 2’-deoxy-D-ribose as its

sugar, while RNA consists of ACGU and the sugar of D-ribose.1

While naturally occurring oligonucleotides are typically less than 20 nucleotides

in length, the synthetic oligonucleotides of interest in this research can be greater than ten

times that number in length.2 These synthetic oligomers have found a range of medical

and therapeutic uses, from DNA sequencing to genotyping to functioning as primers in

quantitative polymerase chain reactions.3 Of particular interest to researchers is the in

vivo stability of these polymers due to their chemical modifications, typically

“sulfurization, modification of the deoxyribose ring, or alteration of the entire oligomer

backbone”.4

In particular, this thesis will examine the analysis of antisense oligonucleotides.

“Antisense oligonucleotides are single strands of DNA or RNA that are complementary

to a chosen sequence. In the case of antisense RNA they prevent translation of

complementary RNA strands by binding to them. Antisense DNA can be used to target a

specific, complementary (coding or non-coding) RNA. If binding takes places this

9

Figure 1: Nucleic Acid Pictorial

10

Figure 2: Oligonucleotide Synthesis5 Synthesis cycle

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

techniques

using

cleic

9

f

ed,

FIP),

16

dimethylbutylammonium-bicarbonate (DMBA), dimethylbutylammonium acetate

(DMAA), piperidine, and imidazole.4,16,18,19,20 These components have been introduced to

the oligonucleotide assay to create a desired chromatographic result, such as the addition

of TEA to bind the oligonucleotide to the column sorbent and allow controlled elution

with the addition of organic solvent. The use of HFIP as a buffering acid has been found

to improve the performance of TEA, wh

ile the combination of piperidine and imidazole

as been shown to increase the mass-to-charge ratio of measured oligonucleotides,

thereby improving response in the higher ma range.16,20 The compounds DMBA and

DMAA have been used to improve separation between oligonucleotides of similar length,

19

components appear to show improved performance when matched with acetic acid, and

19

two to three minutes, while the salts are washing to waste.11 The polymers are then eluted

11

cleaner source of ions is delivered to the instrument. As these compounds are usually

effective, an optimized method must be both efficient and durable.

Therein lies the focus of this research. PPD currently uses a basic oligonucleotide IP-RP-

t

cleanliness and instrument variability. In order to improve this method, several areas of

h

ss

although DMBA is regarded as better for mass spectrometry. Some of these ion-pairing

increased organic concentration in the mobile phase. Most of the current techniques use

an on-line desalting procedure, which loads the sample under high aqueous flow rate for

from the column with a higher organic flow. By virtue of the desalting procedure, a

extracted from biological matrices, cleaner extracts promote assay health. To be truly

HPLC/MS/MS assay for analysis; however, this assay can exhibit difficulties with extrac

the extraction and instrumentation procedures were investigated and compared against

17

other possible options. By compiling optimized individual investigations, this researc

leads to an overall improved assay.

h

Methodology

techniques were examined using porcine, monkey and mouse tissues. Initially, the buffer

use for these extractions, and they usually have some combination of

the detergent has proved vital for column health, and a varied group of detergents, octyl

(SDS), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and

extraction cleanliness, as this component has previously affected both the extraction and

ples. The evaluation of these buffers relied on

qualitative comparisons of chromatography appearance and column degradation, as well

chloroform solution in a liquid-liquid extraction. As this element of the assay has been

investigated in this research.

During the research of this thesis, several oligonucleotide analytical separation

solution of the extraction was tested. Currently, there are several recommended buffers in

Tris[hydroxymethyl]aminomethane (Tris), Ethlenediaminetetraacetic acid, disodium salt

dihydrate (EDTA), sodium chloride (NaCl), and detergent. Through previous experience,

phenoxylpolyethoxylethanol (Igepal CA-630, NP-40 alternative), sodium dodecyl sulfate

polyoxyethylene (20) sorbitan monooleate (Tween-80), were tested for this experiment.

The initial stages of this investigation determined which buffer best aids in tissue

the polymer stability in stored sam

as quantitative comparisons of average analyte response and detection limit.

The initial extraction step utilizes phenol/chloroform/isoamyl alcohol solution and a

both thoroughly evaluated, and typically unchanged, this stage of the extraction was not

18

Typically, the greatest challenge for high quality tissue oligomer analysis is the

cleanliness of the resulting extracts.21 To this end, solid phase extraction (SPE) testing

using both standard and ion pairing techniques was conducted, on a range of SPE

cartridges of Oasis HLB (Waters, polymeric hydrophilic, lipophilic reverse-phase

sorbent), Oasis WAX (Waters, polymeric weak anion exchange mixed-mode rever

phase sorbent), and Spec Plus 3 Phenyl (Varian, polymeric modified reverse-pha

sorbent with phenyl functional groups. These tests evaluated different SPE technique

ion-pairing wash solutions and elution methods and strength to identify the strongest

candidate for an optimized procedur

se-

se

s,

e. Previous methods utilized the ion-pairing strength

t

ess of

ated the reverse phase LC

conditions of the mass spectrometer assay. A variety of column sorbents were examined,

including C18, Phenyl-Hexyl, and mo eloped, robust sorbents, in the

pursuit of the best option for optimum adsorption. Assuming that the cleanliness of the

extracts has been optimized, smaller micron pore size became a viable option to improve

oligomer separation as well. Mobile phase variations were also examined, as there exists

a wealth of suggestions in previous research. The currently popular TEA/HFIP buffer

conditions were tested against the piperidine/imidazole conditions. Of particular interest

was the possible reduction in the number, and thus the increase in MS response, of the

oligomer charge states found in the MS analysis of these polymers after adjustments to

of TEAA in the SPE procedures, while new literature indicated that TEA and HFIP migh

better create the sorbent binding needed for adequate sensitivity of the extracts.21 Later

testing evaluated the SPE methods for different matrices. In this way, the cleanlin

the extracts was improved.

The final components of the assay optimization investig

re recently dev

19

the pH of the mobile phases used in the assay.20 These charge states could also be

reduced by altering the MS parameters, such as collision energy and ion source

temperature, utilized during the mass spectrometry analysis for the compounds.22 In

addition, with improved separation techniques, the gradients were shortened and sample

analysis efficiency was increased.

Once the extraction was optimized, the assay was extended to plasma and urine. By the

adaptation of this assay to other matrices, the difficulties posed by the variations between

these different biological mediums were examined, and overcome. Through this

streamlining of the oligomer extraction process, a better understanding of the chemical

reactions of the nucleic acids themselves was reached.

Experimental

I. Detergent Determination

To examine the action of this buffer component, identical buffers were prepared with

the standard concentrations of the non-detergents additives. This testing profiled buffers

with Tris (usb), EDTA (Sigma), and NaCl (Sigma), and octyl

phenoxylpolyethoxylethanol (Igepal CA-630, NP-40 alternative, Calbiochem, non-ionic),

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, Sigma,

zwitterionic), sodium dodecyl sulfate (SDS, Sigma, anionic), or polyoxyethylene (20)

sorbitan monooleate (Tween-80, Sigma, non-ionic). Each buffer was prepared in 1000-

mL volumetric flasks with nominal masses of 2.42 g Tris, 7.44 g EDTA, and 5.84 g

NaCl. Five milliliters of NP-40 alternative, 10% SDS in water, and Tween-80,

respectively, were added to the appropriate buffer, while forty-three milliliters of 200

mM CHAPS were added to the CHAPS buffer flask to allow a standard addition of ~ 5.3

20

grams of detergent to each buffer. Each buffer was then brought to volume with WFITM

water. Each of these four homogenate buffers was used to prepare a 0.200 g/mL porcine

enate by combining one gram of porcine liver with four milliliters of buffer,

and

app e es were then spiked to a 2500 ng/g

con e

stan n (Appendix C), and injected using the

orig

olig

ma

II. Initial SPE Examin

To further inve re performed on a

variety of matrix samp

samples, using three ty thout ion-pair binding during solid

phase extraction. The

cartridge, a Waters Oa s WAX 10 mg

cartridge.

Using a porcin uffer containing 20 mM Tris, 20 mM

EDTA, 100 mM NaC ples

were spiked to 2500 n roup) were extracted

as f

Proced

ase Lock Gel tube. Add 25 µL of the working solution into the homogenate tube.

liver homog

then processing with a rotary probe homogenizer at low speed until the homogenate

ear d homogeneous (~2-5 min). These homogenat

centration with the target oligonucleotide. These samples were extracted using th

dard liquid-liquid phenol-chloroform extractio

inal parameters of the LC system (Appendix C). As this was the first test in the

onucleotide optimization work, all later improvements to the assay had not yet been

de.

ation

stigate this area, several levels of testing we

les. The initial testing was performed on porcine liver homogenate

pes of cartridges with and wi

three SPE cartridges tested were Spec Plus 3mL PH 15 mg

sis HLB 10 mg cartridge, and a Waters Oasi

e matrix blank made from a b

l, 0.5% NP-40 alternative and adjusted to pH=8.0, 20 blank sam

g/g ISIS 388626. The samples (x4 per SPE g

ollows:

ure:

1. Transfer a 0.100-mL aliquot of blank bulk matrix lysate-homogenate to a Ph

21

2. Add 200 µL of WFITM water to each sample.

4. Add 300 µL of 25:24:1 phenol/chloroform/isoamyl alcohol to each sample.

6. Centrifuge at 15000 rpm for five minutes.

8. Gently rock each tube several times, mix vigorously for thirty sec

3. Add 100 µL of concentrated ammonium hydroxide to each sample.

5. Gently rock each tube several times and then mix vigorously for thirty seconds.

7. Add 300 µL of chloroform to each sample. onds, and

centrifuge at 15000 rpm for five minutes. 5, add 75 µL of 1 M TEAA. Transfer the aqueous extract

the appropriate SPE filtration tube during the load step. 10.

i. Group 1: Condition a Strata Phenyl cartridge with 1.0 mL

Wash with 1.0 mL 0.1 M TEAA, and 1.0 mL water. Elute with 1.0

ii. Group 2: Condition an HLB 10 mg cartridge with 1.0 mL with

1.0 mL 2% Formic Acid, and 1.0 mL water. Elute with 1.0 mL

iii. Group 3: Condition an HLB 10 mg cartridge with 1.0 mL


iv. Group 4: Condition a MAX 10 mg cartridge with 1.0 mL

1.0 mL 2% Formic Acid, and 1.0 mL water. Elute with 1.0 mL

v. Group 5: Condition a MAX 10 mg cartridge with 1.0 mL


11. Evaporate under a nitrogen stream at approximately 45 °C. and mix vigorously for 30

sec. ing tests conditioned the cartridge with methanol and water prior

to load

9. For SPE Group 1, 3, and

SPE extraction:

methanol, and 1.0 mL 0.1 M TEAA. Load the sample slowly.

mL 1% TEA in 90:10 methanol/water, v/v.

methanol, and 1.0 mL water. Load the sample slowly. Wash

75:25 Methanol/Water, v/v.



methanol, and 1.0 mL water. Load the sample slowly. Wash with

75:25 Methanol/Water, v/v.



12. Reconstitute with 100 µL of WFI water. Seal the plate

The non-ion pair

ing the sample on the SPE bed. The sorbent was then washed with an acidic

aqueous solution and water, and then eluted with 75:25 methanol/water, v/v. The ion-

pairing procedure conditioned the SPE sorbent with methanol and 0.1M TEAA prior to

loading the sample on the SPE bed. The sorbent was then washed with water and a 0.1M

TEAA solution, and eluted with 1% TEA in 90:10 methanol/water, v/v.

22

III. Mobile Phase Components

To test the performance of various mobile phase compositions and conditions, an

oligonucleotide solution was prepared at a concentration of 2500 ng/mL in WFITM water,

and injected with the mobile phases over a range of variables, including different ion-pai

reagents, strengths, and pH ranges, on a Phenomenex Phenyl-hexyl 2 x 50, 3 µm,

column. Each mobile phase was teste

r

d with three injections of the high-level external

parameters listed in Appendix C. The tested mobile phases were

water a

n used

high level external under the LC parameters listed in Appendix C.

-4337-B0); Phenomenex

r-RP, 50 x 2 mm, 2 µm (Part No. 00B-4371-B0); Varian Pursuit Diphenyl,

solution, using the LC

nd methanol with ion-pairing concentrations of 0.5:5.0 TEA/HFIP, v/v; 1:10

TEA/HFIP, v/v; 1:10 TBA(tributylamine)/HFIP, v/v; 1:10 TPA(tripropylamine)/HFIP,

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.


24


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

for 2

1, 3: TEA/HFIP method. 2,4: TEAA method with 90% organic elution.

up 3-4: Post-spiked samples. up 5: Blank, Blank with IS, LLOQ samples in both plasma and liver, exups 1,3.

previously tested samples had exhibited poor re

e also collected and analyzed.

. SPE Comparison

As the

wo SPE procedures was initiated. The following procedure was used

nctional method for SPE analysis of the plasma and

omparison to tissue samples extr

ts followed the same procedure, except skipping steps 8-10

25

appropriate well of a 9

thro se Lock gel tubes are

used to

ltrafree filters are used to filter the extract. As plasma samples cannot be injected onto

the system without the purifying benefits of SPE, there were no comparisons with plasma

samples of that nature.

1. Transfer a 0.100-mL aliquot of blank bulk matrix homogenate or plasma to a l tube. Add 25 µL of the analyte working solution into the

homogenate tube (all but post samples). Add 25 µL of IS1 (all but post samples). 2. 3. Add 100 µL of concentrated ammonium hydroxide to each sample.


7. Add 300 µL of chloroform to each sample.

centrifuge at 15000 rpm for five minutes.

10. Transfer the aqueous extract to the appropriate SPE filtration tube during the load

11. SPE extraction:

mL MeOH and 1.0 mL 8 mM TEA/100 mM HFIP in water.

BLOWDOWN. Load the sample slowly. “Elute” with 1.0 mL

ii. Hydro-Clean Method: Condition an HLB 10 mg cartridge with 1.0 TEA/WFI Water/Methanol, v/v/v, 1.0 mL

methanol, 1.0 mL water and 1.0 mL 0.1 M TEAA. Load the slowly. Wash with 1.0 mL of water (4x). Elute with 1.0 mL

90:10 Methanol/Water, v/v. 12.13. Reconstitute with 100 µL of WFI water. Transfer extracts to a plastic well in a 96-

for 2 minutes.

The IP filtration method and the more standard Hydro-Clean method were used to

extract a series of matrix samples, which were then evaluated for average analyte

6-well plate prior to step 11; these extracts were also filtered

ugh a 0.22 µm Ultrafree filter (Millipore) prior to injection. Pha

separate aqueous and organic layers during liquid-liquid extraction, while

U

Procedure:

Phase Lock Ge

Add 200 µL of WFI water to each sample.

4. Add 300 µL of 25:24:1 phenol/chloroform/isoamyl alcohol to each sample.


8. Gently rock each tube several times, mix vigorously for thirty seconds, and

9. Add 100 µL of 1 M TEAA to samples.

step.

i. IP Filtration Method: Condition an HLB 10 mg cartridge with 1.0

CATCH LOAD AND ELUTION VOLUMES FOR

70:30 MeOH/8 mM TEA, v/v.

mL of 1:10:90

sample

Evaporate under a nitrogen stream at approximately 45 °C.

well plate. Seal the plate and mix vigorously for 30 sec, then centrifuge at 3500

26

response and column effect (i.e., damage to the column caused by the lack of cleanliness

of the extracts).

VIII. Urine Analysis

Urine sample analysis was tested using the oligonucleotide assay. These samp

were extracted with both the standard liquid-liquid extraction found in Appendix C (w

and with

les

ith

out Ultrafree filtration) and the liquid-liquid extraction with the addition of the

cedure, as listed in the previous section. As any investigation of

urine e

sted

f guard columns were examined. A

series of guard column were tested for analyt analysis. The guard columns were

evaluated for overall peak shape and average response, and were tested with three

injections of tissue sample extract under the LC conditions listed in Appendix D (minus,

of course, the guard column). The guard columns tested were Phenyl-hexyl Guard, 4 x 3

mm (Part No. AJO-4351); Phenyl-hexyl Guard, 4 x 2 mm (Part No. AJO-4350); Polar RP

Guard, 4 x 3 mm (Part No. AJO-6076); AQ-C18 Guard, 4 x 2 mm (AJO-7510); C18

IP Filtration SPE pro

xtraction should also examine any possible binding of the compound to the walls

of the storage unit utilized in the method, oligonucleotide storage adhesion was te

using this assay. A pool of urine was spiked with analyte and left refrigerated at 2-8 °C

for an hour. Samples from this pool were then analyzed by the standard liquid-liquid

extraction found in Appendix C, and compared to samples spiked with analyte

immediately prior to the extraction.

IX. Guard Column Analysis

Although the chromatography used in the previous testing was functional for test

injections, further improvements through the use o

e

27

Guard, 4 x 3 mm (Part No. AJO-4287); Gemini C18 Guard, 4 x 2 mm (Part No. AJO-

7596).

X. Tissue Specifics

When certain tissue types, such as spleen, lung and skin, are extracted, the colum

becomes clogged almost immediately. To alleviate these extraction variations, a

chloroform wash prior to the addition of the ammonium hydroxide in the liquid-liquid

extraction was tested (please see Appendix D, step 4 for extraction details). In addition,

there can be variability between the response ratios of different tissue types. To dilute

these matrix effects, smaller portions of the tissue sample (25-40%) were extracted, to

reduce the matrix effects of the different tissue types. These tests portioned 125-200 µL

of the original 20 milligram / 500 µL sample for extraction (please see step 2 of the tissu

extraction in Appendix D for procedure details).

n

e

Results and Discussion

I. Detergent Determination

Oligonucleotides are extracted out of tissue that has been homogenized in buffer

containing specific components to improve assay viability. One important component of

this buffer is the detergent, or surfactant. Originally used as defense against any

pathogens found in the tissue, subsequent laboratory work conducted on a project

unrelated to this thesis found that it also played an important role in the health of the

column. As this component of the buffer appears to be significant, initial testing was

conducted to determine if the standard buffer detergent used in oligonucleotide work

could be improved.

28

Surfact

s.

ds can

est is most focused on determining if the charge classification of

surfactant in the homogenization buffer will be relevant to its use in the oligonucleotide

assay.

The buffer utilizes Tris (20 mM) and NaCl (100 mM) to promote pH stability, and

EDTA disodium salt (20 mM) as a chelating agent to scavenge metal ions in the

homogenate and deactivate metal-dependent enzymes, which could potentially damage

the oligonucleotides. These compounds are typically dissolved in WFITM water, with

0.5% NP-40 alternative as the detergent, and pH adjusted to 8. This test compared the

behavior of NP-40 alternative to SDS, CHAPS, and Tween-80.

From the test injections, it was clear that all four detergents performed adequately

for column conditioning and ruggedness purposes, as the overall system pressure did not

increase with these injections. Additionally, peak shape was maintained fairly well

among all detergent candidates, and all four detergents showed good response (Figures 4-

ants are typically amphiphilic organic compounds, containing both hydrophilic

(“head”) and hydrophobic (“tail”) groups. By this structural mechanism, they are soluble

in both aqueous and organic solutions, and able to bond with both lipids and protein

Surfactents are known to create micelles, with the hydrophobic (lipophilic) ends bonding

to lipids in the homogenate, while the hydrophilic ends of the compounds shield the oil

from the surrounding aqueous solution. These compounds can also form reverse micelles,

with the hydrophilic ends bonding to lipophobic compounds, while the tails shield the

reverse micelle from the surrounding oil. The hydrophilic ends of these compoun

be neutral (non-ionic) or carry a charge (anionic for negative charge, cationic for positive

charge). A zwitterionic surfactant carries two oppositely charged groups on the

hydrophilic head. This t

a

29

7). How

ould

the ithout damage

to the oligonucleotide.

II. Initial SPE Examination

For most documented oligonucleotide work, the preliminary extraction is performed with

a liquid-liquid method utilizing 25:24:1 phenol/chloroform/isoamyl

alcohol. From previous work with these compounds, PPD has added a secondary wash

with chloroform to remove additional lipid molecules from the aqueous extract prior to

evaporation and reconstitution. However, these extracts typically exhibit varying degrees

of matrix suppression from extracted biological components, in addition to compromising

column performance due to sorbent pollution. To better purify these extracts, solid phase

extraction can be added to the assay after completion of the liquid-liquid component. This

addition is particularly useful when attempting low limits of quantitation, as cleaner

extracts provide higher overall response.

Theoretically, the SPE process should remove biological remnants such as protein

nd lipid particles while allowing the analyte to be washed and then eluted off of the

orbent. A normal process uses charged conditions (in this case, acidic) of the sorbent to

electively bind the analyte at will. The ion-pairing procedure will use the ion-pairing

gents (for example, triethylamine, triethylammonium acetate, 1,1,1,3,3,3-hexafluoro-2-

ever, NP-40 alternative showed the highest, most consistent analyte response (see

Table 1), and retains its role as the buffer detergent. Upon review of this data set, it w

appear that the most beneficial detergent has a high micelle molecular weight and median

molar mass (90,000 Da, and 650 amu for NP-40), creating a low critical micelle

concentration without strongly denaturing characterictics. Through these characteristics,

detergent could be an effective inhibitor of biological contaminants w

a

s

s

a

30

Figure 4: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% CHAPS

31

Figure 5: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% NP-40 Alternative

32

Figure 6: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% SDS

33

Figure 7: 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% Tween-80

34

Table 1: Buffer Detergent Data

Sample Name Area CHAPS 1-1 189855 CHAPS 1-2 139977 CHAPS 1-3 151932 Average 160588 StDev 26041 % Coefficient of Variation 16.2 NP-40 Alt 1-1 169182 NP-40 Alt 1-2 171116 NP-40 Alt 1-3 165971 Average 168756 StDev 2599 % Coefficient of Variation 1.54 SDS 1-1 129172 SDS 1-2 155120 SDS 1-3 148430 Average 144240 StDev 13472 % Coefficient of Variation 9.34 Tween-80 1-1 144672 Tween-80 1-2 180346 Tween-80 1-3 145026 Average 156681 StDev 20495 % Coefficient of Variation 13.1

35

propanol) to bind to the sorbent and the analyte, and then be selectively eluted off of the

so analyte.

The resulting data indicated that the HLB cartridges were the most reliable,

ad s SPE sorbent when utilized with an ion-pair procedure (Table 2). These

results are expected, as the HLB sorbent is known for its versatility and durability. HL

is comprised of a divinylbenzene and N-vinylpyrrolidone copolymer, which allows both

versed-phase interactions (due to the divinylbenzene group) and hydrogen bonding (due

to the hydrophilic N-vinylpyrrolidone) between the sorbent and analyte and/or ion-pair

reagent. In addition, this SPE cartridge has a higher sorbent capacity than classic C18

sorbents, and maintains analyte retention after drying.21 WAX cartridges showed no

analyte in the resulting extracts, possibly indicating that hydrogen and/or weak anion

exchange used by this sorbent was not sufficient to create satisfactory SPE analysis for

this small oligonucleotide. The phenyl cartridges showed variable recovery,

nature of the sorbent was partially successful with the

procedu s

ced

nder

rbent with the

vantageou

B

re

demonstrating that the aromatic

re. However, this sorbent is typically used in nonpolar extractions, and is perhap

not to be the expected choice for SPE analysis of this compound. Acidic loading

conditions resulted in no recovery of the analyte from the HLB cartridges.

These results are consistent with the results expected after examining the ion-

pairing strategy used in LC loading and analysis of these compounds, and thus reinfor

a basic concept for oligonucleotide purification. The compounds must be loaded on

appropriate sorbent with ion-pairing reagents to hold fast during washing, and elute u

the appropriate organic conditions. Further investigation of SPE procedures was

conducted at a later point in the research.

36

Table 2: Initial SPE Comparison

Sample Name Area Phenyl 1-1 142964 Phenyl 1-2 15691 Phenyl 1-3 68766 Phenyl 1-4 27704 Average 63781 StDev 57472 % Coefficient of Variation 90.1 HLB Straight 1-1 0 HLB Straight 1-2 0 HLB Straight 1-3 0 HLB Straight 1-4 0 Average 0 StDev 0 HLB IP 1-1 75924 HLB IP 1-2 206005 HLB IP 1-3 193785 HLB IP 1-4 223565 Average 174820 StDev 67054 % Coefficient of Variation 38.4 WAX Straight 1-1 0 WAX Straight 1-2 0 WAX Straight 1-3 0 WAX Straight 1-4 0 Average 0 StDev 0 WAX IP 1-1 0 WAX IP 1-2 0 WAX IP 1-3 0 WAX IP 1-4 0 Average 0 StDev 0

37

III. Mobile Phase Components

Previous work conducted at PPD has always employed mobile phase conditions

with triethylamine (TEA) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The basic

concept of oligonucleotide analysis revolves around ion-pair binding on a liquid

chromatography column. The protonated TEA, a mobile phase component, binds to

sorbent of the column, and subsequently to the analyte as the sample is loaded on the

column. The sample is loaded on to the column during a high aqueous mobi

the

le phase flow

to conc

t,

for

hains. This improved binding

increases the difficulty of separation of oligonucleotide compounds of similar size during

elution, as longer chains require a higher concentration of methanol to break the

TEA/sorbent binding. As all oligos share a daughter ion, and thus a tendency for cross-

analyte interference, chromatographic separation of the compounds becomes very

important in MS/MS analysis.

Oligonucleotide chromatography thereby exhibits a strong dependence on mobile

phase composition and ion-pair binding efficacy. Testing commenced using the current

mobile phase conditions utilized in the oligonucleotide assays at PPD. Mobile Phase A

consists of a 0.5:5:500 TEA/HFIP/WFITM water, v/v/v, solution, which is approximately

7 mM TEA and 100 mM HFIP, while Mobile Phase B is 0.5:5.0:500

entrate the oligonucleotide molecules into a tight band on the column, while

washing extraneous salt and metal ions from the analyte. The gradual addition of

methanol to the mobile phase composition breaks the binding of the TEA to the sorben

and thus elutes the TEA (and thereby the analyte) to the mass spectrometer for analysis.

Each unit of the polyanionic backbone of the oligonucleotide serves as a binding site

the TEA, and thus binding improves for longer oligo c

38

TEA/HFIP/methanol, v/v/v. This mobile phase arrangement resulted in reasonable peak

shape and response (Figure 8). The next tested mobile phases doubled the TEA and HFIP

strength, and resulted in a quadrupling of response and improved column retention, which

could be useful in oligonucleotide separation (Figure 9). Increased ion-pair binding

reagents should increase retention, as the analyte will indicate a higher affinity to the

sorbent with a higher concentration of binding agent present; the results reinforce this

hypothesis.

The next tested mobile phases replaced the TEA in the 1:10:500

TEA/HFIP/Solvent, v/v/v, mobile phases with tributylamine, tripropylamine, and

diethylamine, successively, to test the importance of the TEA compound. None of these

mobile phases resulted in initial retention of the compound during the loading conditions,

although the diethylamine mobile phases did create a unique peak during elution (Figures

10-12). The differences between tripropylamine, diethylamine, and triethylamine likely

result from the elevated strength of triethylamine as a simple, polar, easily substituted

nger ion-pairing

re that the low polarity and steric hindrance of

he next mobile phase conditions tested investigated an imidazole/piperidine ion-

pairing concept found in the literature.20 Although this set of conditions resulted in a

unique analyte peak during elution, roughly half of the compound was lost during the

loading period, resulting in lower overall response of the measured peak (Figure 13). This

research indicates that the current TEA/HFIP pairing results in the strongest column

retention and subsequent elution of the analyte peak.

tertiary amine. As tripropylamine and tributylamine should be stro

agents than triethylamine, it is suspected

these reagents caused reduced performance with the phenyl-hexyl column.

T

39

Figure 8: 0.5:5.0:500 TEA/HFIP/MP, v/v/v

Standard Mobile Phase composition yields reasonable retention and response.

40

Figure 9: 1:10:500 TEA/HFIP/MP, v/v/v

he increased ion-pairing reagent concentration incrT eases oligonucleotide column retention and response.

41

Figure 10: 1:10:500 TBA(tributylamine)/HFIP/MP, v/v/v

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%


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

Mean 1165262 1399469 Mean 1642246 1879283

of Variation 20.7% % Coefficient

27.5% % Coefficient of Variation 11.1% 13.6%

%Recovery 71.0% 74.5% LLE I

FiltratiSPE

P on

Liver

fortified) Response Response fortified) Response Response

Sample ID (pre-

extraction Analyte Int. Std.

Sample ID (post-

extraction Analyte Int. Std.

PRE 1-1 1641824 2166915 Evap 1-1 1788949 2700289 POST Pre-

PRE 1-3 1599430 2043211 POST Pre-Evap 1-3 2234663 3175339

Mean 1654441 2104968 Mean 1980421 2883944

SD 62285 61852 SD 229391 255194

% Coefficient % Coefficient of Variation 3.76% 2.94% of Variation 11.6% 8.85%

.0% %Recovery 83.5% 73

PRE 1-1 1641824 2166915 POST Post-Evap 1-1 2089924 3004655

PRE 1-2 1722069 2104778 Evap 1-2 2004779 2831457 POST Post-

PRE 1-3 1599430 2043211 POST Post-Evap 1-3 1866659 2501978

Mean 1654441 2104968 Mean 1987121 2779363 SD 62285 61852 SD 112675 255356



%Recovery 83.3% 75.7%

66

Table 6 cont. Hydro-Clean SPE Filtration Liver

.

nse

Sample ID (pre-

Sample ID (post-


Analyte Response

Int. Std. Response


Analyte Response

Int. StdRespo

PRE 1-1 2773213 4809971 POST Pre-Evap 1-1 5773569 6916000

PRE 1-2 3342467 4159778 Evap 1-2 5878784 61POST Pre-

49047

PRE 1-3 3066735 3666718 POST Pre-Evap 1-3 5275986 6028030

Mean 3060805 4212156 Mean 5642780 6364359 SD 284674 573423 SD 321980 481552



%Recovery 54.2% 66.2%

67

Table 7: Kidney Recovery

fortified)

Sample ID

Analyte Response

Std. onse

Sample ID (post-

extractifortifie

Analyte Response

Int. SRespo

(pre-extraction Int.

Respon d)

td. nse

LLE 0.22 µm filter

Kidney

PRE 1-1 1232453 POST Pre-Evap 1-1 2103173 1926667 1040685

PRE 1-2 882351 726731 POST Pre-Evap 1-2 1649716 1880779

PRE 1-3 1004510 919097 POST Pre-Evap 1-3 1647032 1707574

Mean 1039772 504 Mean 1799974 1838340 895



%Recovery 57.8% 48.7% SF

PE iltration

fortified) Analyte

Response d. se

ple ID ost-action ified)

Analyte Int. Std. Response

Sample ID (pre-

extraction Int. StRespon

Sam (p

extrfort Response

H

ydro-Clean 0.22 µm

Filtration Kidney

PRE 1-1 687821 321 Pre--1 1626961 1945492 109 0

POSTEvap 1

PRE 1-2 1149569 895 Pre--2 1445460 1044932 117 1 Evap 1

POST

PRE 1-3 540042.80 6.1 Pre--3 908752.83 96967 8

POSTEvap 1 1241159.81

Mean 792477 1080612 1299726 Mean 1437860 SD 317955 105205 SD 193013 563380

fficient iation 13.4% 43.4%


% Coeof Var

overy 55.1% 83.1% %Rec

68

extraction alone, while cynomolgus kidney displayed a recovery of 57.8%. The liver

tissue had a recovery level of 54.2% when treated with the standard Hydro-clean SPE

pro over f 8 h airin ion As

e SPE sample se thro SPE t ther n w

equired for analysis. T a sam hib c of 3 ith

tandard Hydro-clean S E method, and a recovery of 53.2% with the ion-pairing

iltration method. However, there was a loss in rep oduci ility with the ion-pairing

iltration samples, most like the unclean re o ts. W ithe

ese recovery results is optimal for sample analysis, these nu t

provement over the o ethod.

Small batch test was condu fin . It w rmi

at the addition of ion- ing reage e sa r PE p e w

dvantageous, most likely due to the comprehensive exposure to the ion-pairing reagents

necessary for sorbent binding; e additio prove recovery with

e 0.22 µm filtration u id-liquid s with e add

addition, the addition of ion-pairing reagents to the elution solutions

edures improved overall response (Table 9).

lthough the simple injection of the plasma extracts without damage to the

system provement, further testing of the smaller extraction details was

required. As there exists less biological matrix components in plasma extracts, it

appeared that recovery could be further impacted by the loss of analyte exhibited during

extended solvent evaporation with a nitrogen stream. When extracted samples were

compared with blank samples spiked with analyte post-evaporation, recovery numbers

cedure nd a reca y level o 3.5% wit the ion-p g filtrat method. both

of thes ts passed ugh the sorben bed, no fur filtratio as

r he plasm ples ex ited a re overy level 0.4% w the

s P

f r b

f ly due to natu f the extrac hile ne r of

th mbers represent a vas

im riginal m

ing cted to e-tune the extraction as dete ned

th pair nts to th m les pp ior to the S ro edurc as

a

however this sam n did not im

th tilized in liqu extraction conducted out th ition

of SPE (Table 8). In

n both PE proci S

A

was indeed an im

69

dropped to 26.7%. This loss in recovery is hypothesized to be due to an insolubility of the

after e i is not witnessed in tissue

to the m omp

alyte loss, a sm tity ( ) of ethylene glycol was

ach sample prior to eva n. dditio

l t thod (Table 10); the addition of

n p o samp d not prove advisable, as a

se le 11 ll sample run of calibration

controls was extracted with the above method and the run was

2) wi a uracy s set at 25% for the lower

% for r trols were determined to be

mits s % lowe tion and 20% for

er improvem re ple variability would be desirable.

i cond was a great success, both

r v urine at 92.9% when using the

n alone, and w e LLE

(Table 13).

Filtration with 0.22 µm Ultrafree filters was also tested, and while the filtration

was found to be unnecessary for extract cleanliness, assay variability decreased to 1.72

%CV (in comparison to 4.92 %CV without filtration) when the filtration was in place

(Table 14). Thus, the 0.22 µm filtration was retained in the methodology for urine

analysis.

analyte in plasma extracts vaporation. This nsolubility

extracts, theoretically due plentiful atrix c onents found in these extracts.

To alleviate this an all quan 10 µL

added to e poratio This a n improved recovery to 90.4%,

and allowed acceptance of the p asma ex raction me

ethylene glycol to the extractio rocess f tissue les di

drop in recovery sample respon was noted (Tab ). A fu

standards and quality

deemed acceptable (Table1 th calibr tion acc limit

limit of quantitation and 20 all othe levels. Quality con

acceptable with accuracy li et at 25 for the r limit of quantita

all other levels. Furth ents to duce sam

VIII. Urine Analysis

The urine extraction with the liqu d-liquid itions

with and without a SPE procedu e. Reco ery for stood

liquid-liquid extractio 66.9% hen th method was paired with SPE

70

Table 8: Addition of TEAA to LLE Filtration

Area IS Area Ratio_Area Sample Name Liver No TEAA Filtration 1-1 1601237 2566918 0.624 Liver No TEAA Filtration 1-2 1610383 2551677 0.631 Liver No TEAA Filtration 1-3 1472293 2274600 0.647 Liver No TEAA Filtration 1- 4 65 0.617 4 148200 24016Average 0 0.632 152156 2409314 St Deviation 8697 0.015 77076 13% Coefficient of Vari 2.38 ation 5.07 5.76 Liver TEAA Filtra 7 7 1.052 tion 1-1 56610 18913 Liver TEAA Filtra 5 4 1.055 tion 1-2 16041 89348 Liver TEAA Filtra 7 5 1.285 tion 1-3 68775 98123 Liver TEAA Filtra 6 5 1.148 tion 1-4 82433 94495 Average 655750 560655 1.163St Deviation 1 6 28462 61781 0.11% Coefficient of V 9.95 ariation 19.6 11.0

Table 9: Introdu n-pai nts P Loadin During n

e Area IS Area Ratio_ALiver No TEAA Addition Pre 1-1 1487278 2275634 0.654

1450423 2482818 0. 1447740 2582168 0 1570636 2622060 0

e 0 2562349iation 2 6fficient of 1 0

re 1-1 824 2166915 0.758

1722069 21047780 2043211

1618547 1997988Average 1646682 2048659 0.804

iation 3 53603fficient of Variation 1 2

ati e 1435018 1994085

e 5 1985940e 4 1790128e 5 1745476

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%


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

73

Table 12: Plasma Run

Sample Name Area IS Area Ratio_Area Conc. Calc.Conc. Accuracy RB 1-1 0 0 #DIV/0! N/A N/A N/A MB 1-1 0 0 #DIV/0! N/A N/A N/A MB/IS 1-1 0 70072 0 N/A No Peak N/A QC 1-1 9555 62791 0.152 2 2.35 117.74 QC 1-2 9349 61959 0.151 2 2.34 116.86 QC 2-1 1276804 63959 19.963 250 276.67 110.67 QC 2-2 1218464 75975 16.038 250 221.68 88.67 QC 3-1 5166244 93686 55.144 800 784.92 98.11 QC 3-2 3634639 106668 34.074 800 477.12 59.64

Cal 8-1 4243803 95724 44.334 1000 625.67 62.57

curve

Deleted fromcalibration

CB 1-1 6134 0 #DIV/0! 0 N/A N/A CB2 1-1 0 0 #DIV/0! 0 N/A N/A Cal 7-1 4307880 82662 52.114 700 739.99 105.71 Cal 6-1 1228242 76539 16.047 250 221.81 88.73 Cal 5-1 264468 67730 3.905 62.5 53.71 85.93 Cal 4-1 63713 55479 1.148 15 15.96 106.41 Cal 3-1 20071 57698 0.348 5 5.03 100.52

Cal 2-1

Deleted

17088 70123 0.244 2 3.6 180.19

from calibration curve

Cal 1-1 6162 87049 0.071 1 1.24 124.4 QC 1-3 11038 88084 0.125 2 1.99 99.41 QC 1-4 10393 85313 0.122 2 1.94 97.02 QC 2-3 1353336 81846 16.535 250 228.63 91.45 QC 2-4 1298434 90770 14.305 250 197.5 79 QC 3-3 3988205 96996 41.117 800 578.83 72.35 QC 3-4 3332645 97157 34.302 800 480.38 60.05 Cal 8-2 5317167 84042 63.268 1000 906.52 90.65 CB 1-2 7908 0 #DIV/0! 0 N/A N/A CB2 1-2 0 0 #DIV/0! 0 N/A N/A Cal 7-2 4538266 80054 56.69 700 807.92 115.42 Cal 6-2 1317395 77725 16.949 250 234.42 93.77 Cal 5-2 315776 71599 4.41 62.5 60.65 97.04

Cal 4-2 84622 102848 0.823 15 11.51 76.75

curve

Deleted fromcalibration

Cal 3-2 21566 61441 0.351 5 5.07 101.38 Cal 2-2 12487 126060 0.099 2 1.63 81.49 Cal 1-2 6498 109577 0.059 1 1.09 108.73 QC 1-5 14583 117625 0.124 2 1.97 98.5 QC 1-6 10305 63814 0.161 2 2.48 124.1 QC 2-5 1352126 70511 19.176 250 265.62 106.25 QC 2-6 1462637 77154 18.957 250 262.55 105.02 QC 3-5 4942371 75292 65.643 800 942.39 117.8 QC 3-6 5199211 84078 61.838 800 884.99 110.62

74

Table 13: Urine Recovery

ple ID (pre-


Analyte Re Response

le ID (post-

extractionfortified)

alyte sponse

. Std. ponse

Sam

sponse Int. Std.

Samp

AnRe

IntRes

LLE 0.22 µm r filte

Urine PRE 28POST PreEvap 1-1 21-1 3705462 12692

-3532266 456990

PRE 1-2 3134222 7 POST PreEvap 1-2 3776728 2626999 192330

-

PRE 1 19POST PreEvap 1-3 3 2-3 3242238 98285

-545089 502949

Mean 3360641 2244761 Mean 36180 2528979 28

% Coeffiof Variat 9.03% 22.0%


cient ion

t

%Recovery 92.9% 88.8%

PRE POST PoEvap 1-1 5 301-1 3705462 2812692

st-336166 57042

SPE nFiltratio PRE 1

POST Post-Evap 1-2 5165737 3092037 -2 3134222 1923307

PRE 1 19POST PosEvap 1-3 45 27-3 3242238 98285

t-75628 92268

Mean 3360641 2244761 Mean 5025843 2980449

% Coeffiof Variat 9.03% 22.0%


cient ion

t

%Recovery 66.9% 75.3%

75

Table 14: Urine Data

Sample Name Area IS Area Ratio_Area Urine No filter 1-1 4116018 2698103 1.526 Urine No filter 1-2 4115765 2695048 1.527 Urine No filter 1-3 4155522 2720698 1.527 Urine No filter 1-4 3691055 2222374 1.661 Average 3987447 2546040 1.57 St Deviation 257452 280596 0.08 % Coefficient of Variation 6.46 11.0 4.92 Urine Filter 1-1 3705462 2812692 1.317 Urine Filter 1-2 3134222 1923307 1.63 Urine Filter 1-3 3242238 1998285 1.623 Urine Filter 1-4 3153649 1883289 1.675 Average 3176703 1934960 1.64 St Deviation 57580 58377 0.03 % Coefficient of Variation 1.81 3.02 1.72 Filtration lowers response, improves variability Urine 1 hr 1-1 2861034 1822705 1.57 Urine 1 hr 1-2 3151795 1748782 1.802 Urine 1 hr 1-3 3256400 1812328 1.797 Urine 1 hr 1-4 3324808 1965963 1.691 Average 3244334 1842357 1.76 St Deviation 87135 111661 0.06 % Coefficient of Variation 2.69 6.06 3.56 Urine 0 hr 1-1 3371081 1953385 1.726 Urine 0 hr 1-2 3453597 1968220 1.755 Urine 0 hr 1-3 2912688 1604371 1.815 Urine 0 hr 1-4 3180296 1771397 1.795 Average 3182194 1781329 1.79 St Deviation 270459 182127 0.03 % Coefficient of Variation 8.50 10.202 1.71 60 minutes in plastic does not lower response

76

A full sample run of calibration standards and quality controls was extracted with

n

quirements as the plasma assay. This method seems fully applicable to urine analysis

able 15).

In addition, oligonucleotide storage adhesion was tested using this assay. There

as no loss in overall analyte response due to the delay between analyte addition and

xtraction, with the 0 hour test samples averaging 3244334 cps and 1 hour test samples

veraging 3182194 cps, with response ratios almost identical. This lack of variation

dicates that the compound is not adhering to the walls of the polypropylene container.

esting would be conducted over a longer time period for any official assay viability

able 14).

As the binding mechanism for oligonucleotide analysis in LCMS/MS analysis

relies on little to no movement through the column during high aqueous flow, and full

elution once the appropriate level of organic flow has been reached, loading the sample

on a guard column is comparable to loading on an analytical column. Smaller pore size

has proved advantageous in analytical columns, but smaller dimensions appeared to be

less helpful in guard columns. Guard columns were tested at 4.0 x 2 mm and 4.0 x 3.0

mm in size, and the majority of the smaller guard columns achieved insufficient banding

of the target compound. Instead, these smaller guard columns produced a jumbled mass

of compound response, while the larger guard columns produced more reliable peaks at

elution, most likely due to their larger surface area for analyte binding.

However, performance appeared to be just as dependent on sorbent composition.

the above method and the run was deemed acceptable, with the same quantitatio

re

(T

w

e

a

in

T

(T

IX. Guard Column Analysis

77

Table 15: Urine Run

Sample Name Area IS Area Ratio_Area Conc. Calc.Conc. Accuracy RB 1-1 0 0 #DIV/0! 0 N/A N/A UB 1-1 0 0 #DIV/0! 0 N/A N/A UB/IS 1-1 0 1969843 0 0 N/A N/A QC 1-1 6396 1867841 0.003 4 4.64 116.03 QC 2-1 75243 1850947 0.041 50 56.82 113.64 QC 3-1 1114252 1714966 0.65 800 855.04 106.88 Cal 8-1 1437945 1806847 0.796 1000 1033.22 103.32 CB 1-1 564 0 #DIV/0! 0 N/A N/A CB2 1-1 0 0 #DIV/0! 0 N/A N/A Cal 7-1 434041 2326793 0.187 250 257.18 102.87 Cal 6-1 135422 1852220 0.073 100 101.96 101.96 Cal 5-1 69125 1790864 0.039 50 53.96 107.91 Cal 4-1 18114 1936008 0.009 12.5 12.99 103.89 Cal 3-1 7366 1963081 0.004 5 5.1 102.06 Cal 2-1 5628 1885478 0.003 4 4.02 100.57 Cal 1-1 2936 1978734 0.001 2 1.91 95.44 QC 1-2 6431 2123036 0.003 4 4.08 102.12 QC 2-2 69812 1956856 0.036 50 49.87 99.75 QC 3-2 1076444 1902908 0.566 800 750.45 93.81 Cal 8-2 1328913 1793490 0.741 1000 966.84 96.68 CB 1-2 305 0 #DIV/0! 0 N/A N/A CB2 1-2 0 0 #DIV/0! 0 N/A N/A Cal 7-2 424898 2424019 0.175 250 241.95 96.78 Cal 6-2 135065 1943008 0.07 100 96.97 96.97 Cal 5-2 68312 1996782 0.034 50 47.83 95.65 Cal 4-2 17071 1960406 0.009 12.5 12.07 96.59 Cal 3-2 7217 1993546 0.004 5 4.92 98.34 Cal 2-2 5697 1994781 0.003 4 3.84 96.02 Cal 1-2 3188 1960694 0.002 2 2.11 105.47 QC 1-3 5428 1908352 0.003 4 3.83 95.63 QC 2-3 66701 1876308 0.036 50 49.7 99.39 QC 3-3 963126 1833191 0.525 800 699.72 87.46

78

The 4.0 x 2 mm AQ-C18 guard produced no analyte peak at all, merely a mass of

ound at the expected retention time (Figure 25). The 4.0 x 3 mm C18 guard column

xhibited more of a peak, but the peak was jagged and wide, and deemed insufficient for

cceptable chromatography (Figure 26). The 4.0 x 2 mm Gemini C18 guard achieved a

sponse of roughly 4 x 104 cps height, but still displayed a mass of compound in a

mble at the base of the peak (Figure 37). These results mimic the lowered response and

hromatography of the C18 sorbent in the tested analytical columns. The 4.0 x 3 mm

olar RP guard achieved a response of roughly 2 x 104 cps height, but left a mass of

ompound in a jumble at the base of the peak (Figure 28), most likely due to insufficient

urface area for banding. The 4.0 x 2 mm Phenomenex Phenyl-hexyl guard displayed a

gged peak of 1.1 x 105 cps height, but the jagged nature of the peak was deemed

phy (Figure 29); this guard column also appeared

to have insufficient surface area for banding, as the sorbent was clearly not the concern.

The clear winner was the 4.0 x 3 mm Phenomenex Phenyl-hexyl guard column, which

displayed a strong, sharp peak with acceptable chromatography and response (Figure 30).

This result matched that from earlier tests of analytical columns with various

sorbents, and reinforces the chromatographic improvements expected of the phenyl-hexyl

sorbent. As the surface area available for analyte ion-pair binding appeared to be

inadequate, some of the other guard columns may have been sufficient with a sample of

lower concentration.

In addition to improving overall chromatography, this series of successful testing

was relevant in assay ruggedness as well. Previous examinations of tissue extracts

resulted in poor column endurance, as each column would produce variable

comp

e

a

re

ju

c

P

c

s

ja

insufficient for acceptable chromatogra

79

Figure 25: AQ-C18 Guard, 4 x 2 mm

This guard column produces jagged peak shape and unusable chromatography.

80

Figure 26: C18 Guard, 4 x 3 mm

This guard column produces jagged peak shape.

81

Figure 27: Gemini C18 Guard, 4 x 2 mm

This gua

rd produces reasonable peak shape, with some bunching of analyte response at the bottom of the peak.

82

Figure 28: Polar RP Guard, 4 x 3 mm

This guard produces reasonable peak shape, with lots of bunching of analyte response

at the bottom of the peak.

83

Figure 29: Phenyl-hexyl Guard, 4 x 2 mm

This guard produces reasonable peak shape, with some tailin d bunch f analyt

se at the bott the p

g an ing o e respon om of eak.

84

Figure 30: Phenyl-Hexyl Guard, 4 x 3 mm

This guard produces good peak shape, with little to no bunching of analyte response at the base of

the peak.

85

chromatography and increasing LC pressure after 200-300 injections. With the addition

f a guard column to the instrument conditions, the overall assay became easier to

ew guard column cou reasonably be installed prior to every se

s this assay is already igh end of time and resource consumpti

e much appreciated.

tant consideration f e analysis is the to extract numero

f tissue. This was previously ex d with plasma an extracts, but can

different organ types. As oligonucleotides accumulate in many

f the body, the abilit ptably analyze the oligo content of these

tissue types provides a relevan rstanding of the pathways these compoun

xit from their target

ver tissue is generall htforward extra the type listed

other tissue types can cause pr when not specifically addressed during

organ types can

here appears to be a correlation between a higher fibrous content of the tissue and a

extraction. When the varying tissue types of interest for this compo

and kidney tissues d similar, althou identical, response

behavior of brain t ed more sim o that of kidney

ed to be the mos blematic of the original testing, as overall

sue type was higher than the comparable tissues, disrupting the

ed for quantitation (Tables 17-18). The behavior of this analyte in

s than optimum in t iginal batch of testing. An additional

o

maintain, as a n ld more ries

of injections. A on the h on,

thrifty alternatives ar

X. Tissue Specifics

An impor or sampl ability us

types o amine d urine

also be relevant with

different areas o y to acce

various t unde ds

utilize for entry and e cells.

While li y a straig ction of

above, oblems

extraction. Problematic include brain, lung, skin, and spleen tissues.

T

more problematic und

were tested, liver displaye gh not

(Table 16), while the issue seem ilar t

tissue. Lung tissue prov t pro

response in this tis

analyte/IS ratio us

target tissues was les he or

86

Table 16: Tissue Comparison Between a Kidney Calib ion Curve and Kidney an Liver QCs

IS Area rea Conc. lc.Conc. Accurac

rat d

Sample Name Area Ratio_A Ca y RB 1-1 0 0 V/0! A N/A #DI 0 N/TB 1-1 0 0 V/0! A N/A #DI 0 N/TB 2-1 0 0 V/0! A N/A #DI 0 N/TB/IS 1-1 0 716054 0 0 A N/A N/TB/IS 2-1 0 771182 0 0 A N/A N/KQC 1-1 2103 719273 0.003 4 3.19 79.65 LQC 1-1 1656 622823 0.003 4 2.88 72 KQC 2-1 34046 771281 0.044 50 51.16 102.32 LQC 2-1 29226 757543 0.039 50 44.67 89.34 KQC 3-1 495621 804801 0.616 800 758.03 94.75 LQC 3-1 441304 720494 0.613 800 753.66 94.21 Cal 8-1 367448 463547 0.793 1000 995.18 99.52 CB 1-1 0 0 #DIV/0! 0 N/A N/A CB2 1-1 0 0 #DIV/0! 0 N/A N/A Cal 7-1 178067 805771 0.221 250 261.22 104.49 Cal 6-1 75104 872424 0.086 100 100.36 100.36 Cal 5-1 34938 682257 0.051 50 59.43 118.85 Cal 4-1 9000 902933 0.01 12.5 11.36 90.89 Cal 3-1 3289 682585 0.005 5 5.38 107.69 Cal 2-1 3103 821987 0.004 4 4.17 104.34 Cal 1-1 1601 877118 0.002 2 1.91 95.68 KQC 1-2 2687 653441 0.004 4 4.56 114.1 LQC 1-2 2288 790740 0.003 4 3.15 78.8 KQC 2-2 35575 903287 0.039 50 45.61 91.21 LQC 2-2 30784 893461 0.034 50 39.85 79.71 KQC 3-2 727067 1050069 0.692 800 859.47 107.43 LQC 3-2 659691 1202174 0.549 800 670.62 83.83 Cal 8-2 989118 1239002 0.798 1000 1002.9 100.29 CB 1-2 689 3414 0.202 0 N/A N/A CB2 1-2 0 2207 0 0 N/A N/A Cal 7-2 308925 1462717 0.211 250 249.4 99.76 Cal 6-2 88599 1154363 0.077 100 89.37 89.37 Cal 5-2 40169 1088205 0.037 50 42.72 85.45 Cal 4-2 7913 960713 0.008 12.5 9.35 74.8 Cal 3-2 3308 1210460 0.003 5 2.97 59.3 Cal 2-2 4155 1127914 0.004 4 4.07 101.71 Cal 1-2 2113 1095823 0.002 2 2.03 101.64 KQC 1-3 2850 946244 0.003 4 3.29 82.22 LQC 1-3 3179 1139888 0.003 4 3.03 75.76 KQC 2-3 45373 1211052 0.037 50 43.37 86.74 LQC 2-3 36518 1037325 0.035 50 40.73 81.46 KQC 3-3 783238 1231591 0.636 800 784.52 98.06 LQC 3-3 618834 1168083 0.53 800 646.14 80.77

87

problem was found in spleen tissue, as the fibrous nature of that particular tissue caused a

the extracts for those samples. These extracts were

e a column beyo

sue, the samples we rea h a c form wash, which

ns the fibrous nature of the tis nd a for c r extracts.

raction of different species can lead to differences in tissue

ehavior. Cynomolgus brain tissue was indeed more similar to kidney tissue, whereas this

t displayed betwe e b d kidney tissue. And none of the

tissues behaved in a similar fashion to those of cynomolgus tissues (Tables 17-18).

is limited in sma als, it is beneficial from a financial

tandpoint to analyze multiple types of organs and species in a common assay with a

rving as a basis for bration standards and quality controls. When

ssues from various organs and species can be matched, the overall assay is thus

Further testing of smaller portions of the original sample size proved to alleviate

cific issues. Succes act ok a ntage (40%) of the original

and the extraction displaye ssu cific ior (Table 19). However,

ed, the matrix com nents he tis xtraction provide a

the extraction. W a smaller percentage (20-30%) of the tissue

for extraction, va in and ssay became unacceptable.

e correct balance ix reduction and extract stabilization, the tissue

ted out of the as

column deteriorating characteristic of

found to damag nd repair after less than ten injections. In order to

eliminate this is re pre-t ted wit hloro

weake sues a llows leane

In addition, ext

b

similarity was no en mous rain an

mouse

As tissue availability ller anim

s

proxy matrix se the cali

these ti

improved.

these tissue spe sful extr ions to perce

sample, d less ti e –spe behav

as previously mention po of t sue e

stabilizing benefit to hen

sample was taken riability creased the a

However, with th of matr

differences were dilu say.

88

Table 17: Tissue Comparison (Low Le el)

Area Ratio_Area

v

Sample Name IS Area Cyno Brain QC 1-1 .5 1454 0.438 136470 31Cyno Brain QC 1-2 4 4427 0.39 16173 41Cyno Brain QC 1-3 160052.3 8692 0.412 38Average 1 2 1524 0.413 5275 37StDev 14125 0.024 53590 % Coefficient of Variation 9.2 14.4 5.8 Comparison to Cyno Kidney 16.4 23.8 -6.8 Comparison to Cyno Liver .9 -1.3 9.7 7 Cyno Kidney QC 1-1 125855.9 3499 0.415 30Cyno Kidney QC 1-2 138348.2 1690 0.529 26Cyno Kidney QC 1-3 .5 4979 0.387 129522 33Average 2 0056 0.444 13124 30StDev 6421 0.075 36765 % Coefficient of Variation 4.9 12.3 17.0 Comparison to Cyno Liver -7.3 -20.3 17.8 Cyno Liver QC 1-1 130692.1 9718 0.385 33Cyno Liver QC 1-2 143574.4 2724 0.375 38Cyno Liver QC 1-3 150296 406680 0.37 Average 141521 376374 0.377 StDev 9962 33929 0.008 % Coefficient of Variation 7.0 9.0 2.0 Comparison to Cyno Liver 7.8 25.4 -15.1 Cyno Lung QC 1-1 159237 411167 0.387 Cyno Lung QC 1-2 144492.2 412723 0.35 Cyno Lung QC 1-3 128628.2 359443 0.358 Average 144119 394445 0.365 StDev 15308 30322 0.019 % Coefficient of Variation 10.6 7.7 5.3 Comparison to Cyno Kidney 9.8 31.5 -17.7 Comparison to Cyno Liver 1.8 4.8 -3.1 Mouse Brain QC 1-1 187645.6 489448 0.383 Mouse Brain QC 1-2 186160 431891 0.431 Mouse Brain QC 1-3 182301.9 387658 0.47 Average 185369 436332 0.428 StDev 2758 51040 0.044 % Coefficient of Variation 1.5 11.7 10.2 Comparison to Cyno Kidney 41.2 45.4 -3.5 Comparison to Cyno Liver 31.0 15.9 13.6 Comparison to Mouse Kidney 135.1 69.3 42.2 Mouse Kidney QC 1-1 47565 175791 0.271 Mouse Kidney QC 1-2 89332.51 299341 0.298 Mouse Kidney QC 1-3 99648.86 298140 0.334 Average 78849 257757 0.301 StDev 27579 70988 0.032 % Coefficient of Variation 35.0 27.5 10.5 Comparison to Cyno Kidney -39.9 -14.1 -32.2 Comparison to Cyno Liver -44.3 -31.5 -20.1

89

Table 17 cont. Mouse Lung QC 1-1 159938.9 466392 0.343 Mouse Lung QC 1-2 184989.2 459635 0.402 Mouse Lung QC 1-3 187036 430493 0.434 Average 177321 452173 0.393 StDev 15088 19077 0.046 % Coefficient of Variation 8.5 4.2 11.7 Comparison to Cyno Kidney 35.1 50.7 -11.4 Comparison to Cyno Liver 25.3 20.1 4.3 Comparison to Mouse Kidney 124.9 75.4 30.6

90

Table 18

6.9 14.7 8.2idney 15.8 17.3 -0.3

Comparison Cyno Kidney QC 2-1 2081309 269728 7.716

Cyno Kidney QC 2-3 2467686 312614 7.894

StDev 380579 44683 0.110

Comparison to Cyno Liver -14.2 -20.1 7.1

Cyno Liver QC 2-1 2914936 384485 7.581

Cyno Liver QC 2-3 2843048 392848 7.237

StDev 37973 7898 0.232

Comparison to Cyno Liver 16.6 25.1 -6.7

Cyno Lung QC 2-1 2694564 382859 7.038

Cyno Lung QC 2-3 2302339 426901 5.393

StDev 257236 34331 0.823

Comparison to Cyno Kidney 5.3 33.9 -20.9

Mouse BrMouse Brain Q

Comparison to Mouse Kidney 19.9 -15.3 43.1

: Tissue Comparison (High Level)

Sample Name Area IS Area Ratio_Area Cyno Brain QC 2-1 3033417 417665 7.263Cyno Brain QC 2-2 2886101 376673 7.662Cyno Brain QC 2-3 2643286 310282 8.519Average 2854268 368206 7.815StDev 197004 54190 0.642% Coefficient of Variation Comparison to Cyno K

to Cyno Liver -0.6 -6.2 6.8

Cyno Kidney QC 2-2 2842438 359070 7.916

Average 2463811 313804 7.842

% Coefficient of Variation 15.4 14.2 1.4

Cyno Liver QC 2-2 2857781 400273 7.14

Average 2871922 392535 7.319


Cyno Lung QC 2-2 2786775 450500 6.186

Average 2594559 420087 6.206


Comparison to Cyno Liver -9.7 7.0 -15.2

ain QC 2-1 3150864 484033 6.51C 2-2 2991910 392380 7.625

Mouse Brain QC 2-3 2975009 352761 8.433Average 3039261 409725 7.523StDev 97020 67332 0.966% Coefficient of Variation 3.2 16.4 12.8Comparison to Cyno Kidney 23.4 30.6 -4.1Comparison to Cyno Liver 5.8 4.4 2.8

91

Table 18 cont.

Mouse Kidney QC 2-2 2309216 422892 5.461

Average 2535676 483484

Mouse Kidney QC 2-1 2619323 504830 5.189

Mouse Kidney QC 2-3 2678488 522729 5.1245.258

StDev 198339 53232 0.179% CoefficComparison to Cyno Kidney 2.9 54.1 -33.0

Mouse Lung QC 2-2 3118480 442843 7.042

Average 3004176 423475 7.092


ient of Variation 7.8 11.0 3.4

Comparison to Cyno Liver -11.7 23.2 -28.2

Mouse Lung QC 2-1 2783753 406040 6.856

Mouse Lung QC 2-3 3110296 421542 7.378

StDev 190936 18477 0.265

Comparison to Cyno Kidney 21.9 34.9 -9.6Comparison to Cyno Liver 4.6 7.9 -3.1Comparison to Mouse Kidney 18.5 -12.4 34.9

92

Table 19: 40% Matrix Portioning Extraction

Sample Name Area Ratio_Area IS Area Cyno Kidney QC 1-1 742129 0.052 38601 Cyno Kidney QC 1-2 41185 759715 0.054 Cyno Kidney QC 1-3 37015 722213 0.051 Cyno Kidney QC 1-4 39249 760517 0.052 Mouse Kidney QC 1-1 41348 817592 0.051 Mouse Kidney QC 1-2 41992 820571 0.051 Mouse Kidney QC 1-3 40613 785587 0.052 Mouse Kidney QC 1-4 41696 775210 0.054 Cyno Liver QC 1-1 42371 815868 0.052 Cyn r QC 1-2 41199 77o Live 8405 0.053 Cyn er QC 1-3 41271 74o Liv 8620 0.055 Cyn er QC 1-4 41647 7910o Liv 42 0.053 Mouse Liver QC 1-1 41947 821162 0.051 Mouse Liver QC 1-2 41072 807813 0.051 Mouse Liver QC 1-3 40833 766558 0.053 Mouse Liver QC 1-4 43184 870455 0.050 Cyno Spleen QC 1-1 47112 796891 0.059 Cyno Spleen QC 1-2 47675 689923 0.069 Cyno Spleen QC 1-3 44430 727440 0.061 Cyno Spleen QC 1-4 41705 681050 0.061 Mouse Spleen QC 1-1 38852 614671 0.063 Mouse Spleen QC 1-2 36642 555898 0.066 Mouse Spleen QC 1-3 41118 620917 0.066 Mouse Spleen QC 1-4 40865 631155 0.065 Cyno Kidney QC 3-1 13781598 739311 18.641 Cyno Kidney QC 3-2 13613902 735959 18.498 Cyno Kidney QC 3-3 12905368 686059 18.811 Cyno Kidney QC 3-4 13338989 716907 18.606 Mouse Kidney QC 3-1 13535720 756195 17.900 Mouse Kidney QC 3-2 13687911 750048 18.249 Mouse Kidney QC 3-3 14603039 804333 18.155 Mouse Kidney QC 3-4 14277037 758203 18.830 Cyno er QC 3-1 14694134 78Liv 4 18.692 611Cyno er QC 3-2 14850196 786Liv 375 18.884 Cyno er QC 3-3 14483634 770Liv 054 18.809 Cyno er QC 3-4 15221776 821194 Liv 18.536 Mouse Liver QC 3-1 13280701 766658 17.323 Mouse Liver QC 3-2 13657594 785187 17.394 Mouse Liver QC 3-3 13433472 17.531 766254 Mouse Liver QC 3-4 13672054 767522 17.813 Cyno Spleen QC 3-1 14490789 700811 20.677 Cyno Spleen QC 3-2 16402629 840472 19.516 Cyno Spleen QC 3-3 14914500 738085 20.207 Cyno Spleen QC 3-4 15346397 747643 20.526 Mouse Spleen QC 3-1 14663948 613613 23.898 Mouse Spleen QC 3-2 13900485 579282 23.996 Mouse Spleen QC 3-3 13995021 573244 24.414 Mouse Spleen QC 3-4 12951695 524200 24.708

93

Conclusions

After measured research into the methods of oligonucleotide analysis, an

ptimized method has been compiled. This method improved many areas of the

xtending this assay into the areas of plasma and

urine a ed

to be ex

gents

was tes

available for peak shape and overall response in tissue extraction. The original detergent

of the b

rocedure was added to the method to improve extract cleanliness. While this addition is

ly helpful for assay function with tissue, it was required for plasma extractions to

avoid c

standar compound, but showed

improv

The tis e

matrix

minimi

urine analysis, showing good quantitation and no evidence of analyte-container adhesion.

method

when c ition

of this

o

extraction and LC parameters, while e

nalysis. In order to make these improvements, specific areas of the method need

amined.

The first series of tests involved the extraction parameters. A subset of deter

ted as buffer components in the extraction to determine the best detergent

uffer, NP-40 alternative, appears to be the best option. In addition, a SPE

p

mere

olumn deterioration. The analyte tested appeared too small to benefit from

d SPE procedures due to lack of binding sites on the

ement with increased recovery when the SPE bed was used in a filtration method.

sue method was also improved using a smaller fraction of the sample to alleviat

effects between tissue types and with the utilization of a chloroform wash to

ze the fibrous nature of some tissue types. This assay is also fully compatible with

This research also allowed improvements to the instrument parameters of this

. While the original column was determined to show the best chromatography

ompared with other columns, a guard column comparison resulted in the add

component into the instrumentation. This addition improves assay ruggedness as

94

well as

wear. A

chroma

n SPE step during the extraction of improved the cleanliness of the

xtracts, decreasing the matrix effects of sample injection on the system and allowing

ana

agents to the mobile phases, the resulting increased separation between oligonucleotide

pea

jection times required for this analysis; the use of a guard column offers additional

rotection for the analytical column, allowing less problematic, and thus less time

onsuming, injection. By adding sample portioning and chloroform treatments to the

me ysis of various species and tissue sample

crease the overall satisfaction with this assay.

Although there remains much to be discovered in the world of oligonucleotide

nalysis, these improvements should result in a stronger, consistent assay. In addition,

the w be enjoyed over a range of test matrices, from urine to

g of oligonucleotide analysis by mass

decreasing the expense of the analysis through a reduction in analytical column

lso, the stronger ion-pairing components of the new mobile phase improve

tography and overall assay response.

From the work of this thesis, the oligonucleotide assay appears much improved.

The addition of a

e

lysis of various matrices. After the addition of a higher concentration of ion-pairing

re

ks enhances the identification of these analytes, and can serve to decrease the

in

p

c

thod, a single assay can be used for the anal

types, thereby lowering the cost of analysis. This method optimization should greatly

in

a

se improvements can no

several tissue types. As the understandin

spectrometer grows, these analytical methods will only improve.

95

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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

EDTA

M

LC Pump: HP 1100 Series or Shimadzu LC-10AD VP

nalytical Column: Phenom

olumn Tempera

Pump Program: Gradien

Mobile Phase:

Mobile Phase B, Wash obile Phase:

0.5:5.0:500 TEA/HFIP/Methanol, v/v/v, with 10 µm

EDTA

99

Make Up Flow Mobile Water

Make Up Flow Mobile Methanol

F 0.150 mL/min

d Loop

Injection Volume: 10 µL

L bar

A

Phase A:

Phase B: low Rate:

Injector Loop: Standar

C Pressure: 15-120

Autosampler Wash 1: 90:10 WFI Water/Methanol, v/v

pproximate Run Time: 11 min

oading/Washing Pump Program - Step Table 1: L

Composition Step Total Time (

Flow Rate min) (µL/min) A-Load (%) B-Wash (%)

0 0.0 0.500 95 5 1 2.00 0.500 95 5 2 2.10 0.100 0 100 3 6.20 0.100 0 100 4 6.40 0.500 0 100 5 7.40 0.500 0 100 6 7.70 0.500 95 5

11 11.00 0.500 95 5 Eluting

Composition

Pump Program - Step Table 1:

Step Total Time Flow(min) (µL/min) A (%) B (%)

Rate

0 0.0 0.150 77 23 1 2.00 0.150 77 23 2 6.20 0.150 67 33 3 6.60 0.150 30 70 4 7.70 0.150 30 70 5 8.20 0.150 77 23 6 11.00 0.300 77 23

100

Make-Up Flow Program - Step Table 2:

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

Autosampler: Shimadzu SIL-HTC Cycle: LC-Inj Syringe Standard Loop Samp Volume: 10 µL

105

106

LC Mixing Chamber Volume:

10 µL

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,

v/v/v/v

Make Up Flow Mobile Phase A:

Water

Make Up Flow Mobile Phase B:

Methanol

Flow Rate: 0.150 mL/min

Injector Loop: Standard Loop

Injection Volume: 10 µL

LC Pressure: 15-120 bar

Autosampler Wash 1: 90:10 WFI Water/Methanol, v/v

Approximate Run Time: 11 min

107

Loading/Washing Pump Program - Step Table 1:

Composition Step Total Time (min)

Flow Rate (µL/min) A-Load (%) B-Wash (%)

0 0.0 0.500 100 0 1 2.00 0.500 100 0 2 2.10 0.100 0 100 3 6.20 0.100 0 100 4 6.40 0.500 0 100 5 7.40 0.500 0 100 6 7.70 0.500 100 0

11 11.00 0.500 100 0 Eluting Pump Program - Step Table 1:

Composition Step Total Time (min)

Flow Rate (µL/min) A (%) B (%)

0 0.0 0.150 77 23 1 2.00 0.150 77 23 2 6.20 0.150 67 33 3 6.60 0.150 30 70 4 7.70 0.150 30 70 5 8.20 0.150 77 23 6 11.00 0.300 77 23

Make-Up Flow Program - Step Table 2:

Composition Step

Total Time (min)

Flow Rate (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 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

an evaluation of extraction parameters and lcms analysis...

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