THAT ARE NOT ALLOLLIKULTTUUS009958363B2 (12 ) United States Patent ( 10 ) Patent No. : US 9 , 958 ,363 B2 Smart et al. (45 ) Date of Patent: May 1 , 2018 (54 ) FLUOROUS AFFINITY EXTRACTION FOR (56 ) References Cited IONIC LIQUID - BASED SAMPLE PREPARATION U . S . PATENT DOCUMENTS 7 ,273 ,587 B1 9 / 2007 Birkner et al . ( 71 ) Applicant: Agilent Technologies , Inc. , Loveland , 7 ,273 ,720 B1 9/ 2007 Birkner et al. CA (US ) ( Continued ) ( 72 ) Inventors: Brian Phillip Smart, San Jose , CA (US ); Brooks Bond - Watts , Fremont, FOREIGN PATENT DOCUMENTS CA (US ) ; James Alexander Apffel, Jr ., WO WO2007110637 10 /2007 Mountain View , CA (US ) WO WO2011155829 12 / 2011 ( 73 ) Assignee : Agilent Technologies, Inc ., Santa Clara , OTHER PUBLICATIONS CA (US ) Yuesheng Ye, Yossef A . Elabd “ Anion exchanged polymerized ionic ( * ) Notice : Subject to any disclaimer, the term of this liquids: High free volume single ion conductors ” Polymer 52 ( 2011 ) patent is extended or adjusted under 35 1309 - 1317 , plus supplementary data ( pp . 1 - 6 ) . * U . S . C . 154 (b ) by 168 days . (Continued ) ( 21) Appl. No. : 14 /724 ,656 Primary Examiner — Christine T Mui ão( 22 ) Filed : May 28 , 2015 Assistant Examiner - Emily R . Berkeley (6565 ) Prior Publication Data (57 ) ABSTRACT US 2015 /0369711 A1 Dec. 24 , 2015 A method for removing an ionic liquid from an aqueous Related U . S . Application Data sample is provided . In some embodiments , the method includes : ( a ) combining an aqueous sample including an ( 60 ) Provisional application No . 62/ 016 ,000 , filed on Jun . ionic liquid with an ion exchanger composition including an 23 , 2014 , provisional application No . 62 /016 , 003 , ion exchanger counterion to produce a solution including a (Continued ) fluorous salt of the ionic liquid , where at least one of the ionic liquid and the ion exchanger counterion is fluorinated ; (51 ) Int . Cl. ( b ) contacting the solution with a fluorous affinity material, GOIN 1 / 00 ( 2006 . 01 ) thereby removing fluorous salt from the solution and pro GOIN 1 / 34 ( 2006 . 01 ) ducing an aqueous eluate ; and ( c ) collecting the aqueous (Continued ) eluate . In certain embodiments, the method further includes: (52 ) U .S . CI. contacting a cell with an ionic liquid composition to lyse the CPC ...... GOIN 1 / 34 (2013 .01 ) ; B01D 11/ 04 cell and produce an aqueous sample ; and contacting the ( 2013 .01 ); GOIN 1/ 405 ( 2013. 01 ) ; GOIN aqueous sample with a reverse phase substrate , thereby 1 /4055 (2013 .01 ) ; adsorbing proteins and / or lipids of the cell on the substrate. (Continued ) Compositions, kits and systems for practicing the subject ( 58 ) Field of Classification Search methods are also provided . CPC ...... GOIN 1 / 34 (Continued ) 15 Claims, 14 Drawing Sheets

Ion Exchanger Teg. LiNF ,

Hydrophilic IL A Hydrophilic Aqueous sample metabolites : Organic Lipids Hydrophetic 1L w hydrophilic analytes Alkyny! solid phase , Could be simple alkyre accorctyne.

to go N . Azide -containing Solid phase alkyne , Remaining ionic liquid renoved , i ionic liquid . shown as cyclooctyne . front aqueous layer. US 9, 958 , 363 B2 Page 2

Related U . S . Application Data 2009 /0004048 AL 1 /2009 Elliott et al. 2011/ 0124034 A15 / 2011 Kuehnle et al . filed on Jun . 23 , 2014 , provisional application No . 2011/ 0192793 A1* 8/ 2011 Chew .. C12N 1 /06 62 /049 ,285 , filed on Sep . 11 , 2014 , provisional ap 210 /633 plication No . 62 /051 , 804 , filed on Sep . 17 , 2014 . 2014 /0273080 A1 9 /2014 Apffel , Jr. (51 ) Int . Cl. OTHER PUBLICATIONS B01D 11 /04 ( 2006 .01 ) Wei Ren , Aaron M . Scurto “ Phase equilibria of imidazolium ionic GOIN 1 / 40 ( 2006 .01 ) liquids and the refrigerant gas , 1 ,1 , 1, 2 - tetrafluoroethane (R - 134a )” (52 ) U . S . CI. Fluid Phase Equilibria 286 ( 2009 ) 1 - 7 . * CPC ...... Y10T 436 / 145555 ( 2015 .01 ); Y10T Mark B . Shiflett and A . Yokozeki “ Liquid - Liquid Equilibria of 436 / 147777 ( 2015 .01 ) ; Y1OT 436 / 24 ( 2015 .01 ) Hydrofluoroethers and Ionic Liquid 1- Ethyl- 3 -methylimidazolium (58 ) Field of Classification Search Bis ( trifluoromethylsulfonyl ) imide” J. Chem . Eng. Data 2007 , 52 , USPC ...... 436 / 178 2413 - 2418 . * Lu , et al. , “ A Bioelectrochemical Method for the Quantitative See application file for complete search history . Description of the Hofmeister Effect of Ionic Liquids in Aqueous Solution ” , J . Phys . Chem ., 2012 , 116 , 11075 - 11080 . ( 56 ) References Cited Rezaee , et al. , “ Determination of organic compounds in water using dispersive liquid - liquid microextraction ” , Journal of Chromatogra U . S . PATENT DOCUMENTS phy A , 1116 , 2006 , 1 - 9 . 8 ,143 ,381 B2 * 3 /2012 Von Hagen ...... CO7K 14 / 705 Shahriari , et al ., “ Role of the Hofmeister Series in the Formation of 530 / 350 Ionic - Liquid - Based Aqueous Biphase Systems” , J. Phys . Chem ., 8 , 211 ,307 B2 7 / 2012 Chew et al. 2012 , 116 , 7252 - 7258 . 2002 /0072619 Al * 6 /2002 Bonrath ...... C07D 311/ 72 Yao , et al. , “ Dispersive liquid - liquid microextraction using an in situ 549 /411 metathesis reaction to form an ionic liquid extraction phase for the 2006 /0178507 A1 * 8 / 2006 Berry ...... C07B 63 /00 preconcentration of aromatic compounds from water” , Anal Bioanal 536 / 25 . 3 Chem . , 2009 , 395 : 1491 - 1502 . 2007 /0093462 A1 * 4 / 2007 Rogers ...... A61K 9 / 143 514 / 184 * cited by examiner U . S . Patent May1, 2018 Sheet 1 of 14 US 9, 958, 363 B2

Repeat1-4times

CITI ? Moveto secondary extraction

Vortex Spin FIG.1 14

: -??????A??

(1)2 U . S . Patent May 1 , 2018 Sheet 2 of 14 US 9 , 958 , 363 B2 4 Repeat1-4times

Moveto secondary extraction

FIG.2 Vortex Spin

=-4--

(2)

N-> U . S . Patent May 1, 2018 Sheet 3 of 14 US 9 , 958 , 363 B2

hings

the

balance

bilan

Ag®Agvb

MAAAAAAAAAAA

FIG . 3 U . S . Patent May 1, 2018 Sheet 4 of 14 US 9 ,958 ,363 B2

Yeast cells Yeast cells 1000L water 500L water 50 UL HMIM - CI FIG . 4 U . S . Patent May 1, 2018 Sheet 5 of 14 US 9 , 958 , 363 B2

10% ?

funt 30%

EndogenousATPat60min YYYYYYYYYYYYYYYYYYYY 40% HMIM-CIaqueoussolutions 50%

70%

(wn ) suopequeouos diV FIG.5

muleren ondoren Z ZZZZZZ .L€ U ZZIZIIDZZZZZZZZZZ CIHMIM-%ATPin80Endogenous EZZ77777777 RT

Kagamanya ZZZZZZZZZZZZZZZZZZZZ f4bulanONUW062099OO funn y be

-- - Jewe l d / morn www A ZZITIILIZOZITO w ÖLE : (wn ) uopejjuaquoo di U . S . Patent May 1, 2018 Sheet 6 of 14 US 9 , 958 , 363 B2

Metabolism stays quenched using C18 column 00 230 min 2260 min Overnight Overnight2

ATPconcentration(UM)

0 .10 0 Dilute to 10 % (5X ) C18 Cleanup - 1 C18 Cleanup - 2 FIG . 6 U . S . Patent May 1, 2018 Sheet 7 of 14 US 9 , 958 , 363 B2

womenin

centerandfinit

*

*

o

0.05101520253035404550556065707580859095 Countsvs.AcquisitionTime(min) FIG.7A

.

writualesdeinteresti

wan wanne wwwww ini moment 0000 TOOTVO0010 NOQOTOQOTNOOT VOOTVO COCOOOOOLLOW NOON U . S . Patent May 1 , 2018 Sheet 8 of 14 US 9 ,958 ,363 B2

-

.

- ~

~ 5.75{}8519

*

.

~

: * •••••••• .

*

••• ~

=

=

~

=

- .•

~ - * - = *

- ~ - •••

• +-

• Countsvs.AcquisitionTime(min) ~ - ??????? FIG.7B -

- ••••••• -

- }.31352{2503035445556?1,

+

- + -- -

•••••••••••• +

~ +

- +

~ =

- ••••••

= ~ _

"

" - - - -~ _*

-

- - ~ _

"

_ *~

_

_

* " ~ ????????????????????

.

. + .

- - =

= - - - . {}. - -+ + -

.

+ •••••••••••••••• - •••••••••••••• {2 }{ !. { 3 } ?\ \ ' ( V } } } { 4 } \ , } { \ \ . 50.}4. ]_ ? .{_ ,} N N { } } N { { ~ ~ ~ ~ ~ . 3.1_2.}{ 2. 2-15 , }{ 85. ".5~ ~55.{ {3.45- (????? ????????? 1.554 ,1 1.65 1.35 U . S . Patent _ May 1 , 2018 Sheet 9 Of 14 US 9 , 958, 363 B2

????????? 19

8?'6 ???

5'?3.2 Countsvs.AcquisitionTime(min) .'000

08?.Q

6

6 : 0

????0 03 02 1 . 3 ?????????? -??????????

-

75 25 50. b - 85.f 1.65 4.65 45?' 4.35 , Q5,? 95 0 085 0.55 , 25,8 _6. ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????" 35,8_ 2 235. 4 15,1? 065 0 ????? 215F2 ?? ???8. ? ? 20520 5; ??????0 ?3,} 2 x 4 ???????28 ?'c - 32 7 - 3 ( A + ?? 00 } } } 9195 ???? ??? ??? ??? ??? ? U . S . Patent May 1, 2018 Sheet 10 of 14 US 9 ,958 , 363 B2

0.123456789 Countsvs.AcquisitionTime(min)

1.65 45.1 1.25 1.151 1.05 0.95 0.85 0.75 0.651 0.55 0.451 0.35 351.2325.2 -2.15. 2- 1.2 05.2 _ 0. 2 95 .1 .9 .1 851. _8.1 75 1. 7.1 55.1-5.1 Winn 257.0_2.015WUUUUUUUUUUUUU.0 .7. 0 .0 O ping 1.0 0.7 0.5 0.4 ??? ??? ???? ??? U . S . Patent May 1 , 2018 Sheet 11 of 14 US 9 ,958 , 363 B2

omme **** * * * * * ídíníkinu Putus FIG.9 **** * * * * * ** * ** * * * * * * : 17 tudbudististatistiek*** * ** * * pressivement c statuty SEEEEEEEEEEE84930307777777777 777771Fit144444444444444444444444444* ** * * * * * * * * * * * * Sustitu Aqueous-Contains Hydrophobic lonicLiquid Why Metabolites **** * * * * * * * 2 = 444444 4444444444444 *22* ** # 44444444444444 4444444444444444?? 4444444446666 144444 A

trotor 44444444444444** * ** 4777777770h*** * * * * * ** *

diététicahet ## ## #?? ### ???? / etterret44444444444444444*** * * * * * ** * * * ** * * * Mediaremoved typesetastih++++ + 44444444444 elevision 7ttttttttttttttttttty FA44444444444444:77 * * * * * *

77777 **** * * * ptthae ** ** * * * * * * * * * * A 44444444444444444$ 44442#*4444444* * * * * * * 1777777*** * 747971*** * * 4 Cells Filter nearlyall CultureMedium Extracellular Components CellPelletwith liquidremoved U . S . Patent May 1 , 2018 Sheet 12 of 14 US 9 , 958 ,363 B2

3 \ . ?? ? FG . 10 U . S . Patent May 1, 2018 Sheet 13 of 14 US 9 ,958 , 363 B2

lon Exchanger e . g . LINF2

+9+ + + h 91* 11 :*** * * * * * * Hydrophilic IL *** Hydrophilic within0 *** * * * * * * * * * ** Aqueous sample 0 metabolites **- * - 44444450+ + + + ++ + + + + + + Organic Lipids Hydrophilic IL W / hydrophilic analytes **** * * * * * 1404144dusts Alkynyl solid phase , Could be wySorry simple alkyne or cyclooctyne . por

** * *** * * * * * *** * * * * ** * * * * * * * ** * ** *1111111stiaan** * - - ** * * * * + 91977 trítetasiga+35mm

manchaHann VRBr Azide -containing Solid phase alkyne , Remaining ionic liquid removed ionic liquid . shown as cyclooctyne . from aqueous layer. FIG . 11 U . S . Patent May 1 , 2018 Sheet 14 of 14 US 9 ,958 , 363 B2

FIG.12 (6) Aqueous-Contains Metabolites Hydrophobic RonicLiquid TE

thATE Mediaremoved 090

JOOC

0000 : 00000Ugco Cells Filter nearlyall CultureMedium Extracellular Components CellPelletwith liquidremoved US 9 , 958 , 363 B2 FLUOROUS AFFINITY EXTRACTION FOR 50 uL fluorous solvent, 0 .05 equivalents M + -NTfn ; (7 ) IONIC LIQUID -BASED SAMPLE fluorous SPE (maybe not needed ) . PREPARATION FIG . 2 schematically illustrates an ionic liquid metabo lomics sample preparation workflow for methods according CROSS - REFERENCING 5 one embodiment of the subject method : ( 1 ) Fast filter ; ( 2 ) Lyse / Quench 50 uL 1 : 1 HMIMCI:H20 ; ( 3 ) C18 column (remove protein and lipids ); ( 4) Extract HMIMCI 225 uL This patent application claims the benefit of U . S . provi H20 , 100 UL HFE -7100 , 1. 1 equivalents Ag + - - N (perfluoro sional application Ser . Nos . 62/ 016 , 003 , filed on Jun . 23, hexylsulfonyl) 2, (6 ) 50 uL H2O , 50 °L HFE -7100 , 0 .05 2014 , 62 /051 ,804 , filed on Sep . 17 , 2014 , 62 /049 , 285 2 ,3014 filed 10 equivalents Agt- N (perfluorohexylsulfonyl )z ; (7 ) C8 -Fluo on Sep . 11 , 2014 and 62 /016 , 000 , filed on Jun . 23, 2014 , rous SPE (maybe not needed ). which applications are incorporated herein by reference for FIG . 3 shows the structure of a silver salt of an ion all purposes. exchanger of interest (Ag + - NTfn = silver (I ) bis (( perfluoro hexyl) sulfonyl) imide ) . INTRODUCTION 15 FIG . 4 depicts microscope images demonstrating lysis of yeast cells that have been treated with water ( left ) or 1 : 1 Sample preparation is an analytical process which water/ HMIM - C1 ( right ) and stained with trypan blue . Left : includes an extraction procedure that results in the isolation Yeast cells, 100 uL water, add 2 uL to 18 uL Trypan Blue , and enrichment of components of interest from a sample 20x magnification . Right: Yeast cells , 50 uL water , 50 UL matrix . Extraction can vary in degree of selectivity , speed 20 HMIM - C1, add 2 uL to 18 L Trypan Blue , 20x magnifica and convenience and depends not only on the approach and tion . conditions used but also on the geometric configurations of FIG . 5 illustrates the results of an ATP Luciferase assay the extraction phase. There is a constant need for the that demonstrates ATP metabolism in E . coli cells remains development of simplified and miniaturized sample prepa - quenched in aqueous HMIM -Cl solutions. ration methods requiring lower quantities of purification 25 FIG . 6 illustrates the removal ofproteins using a C18 SPE materials and more efficient ways to obtain isolated and (solid phase extraction ) prior to ion exchange reaction keeps purified analytical samples. metabolism quenched . FIGS . 7 A and 7b show extracted ion chromatograms SUMMARY ( EIC ) for HMIM after each extraction of the aqueous layer 30 using fresh HFE - 7100 (methoxyperfluorobutane ) and bis Aspects of the present disclosure include a method for (( perfluorohexyl ) sulfonyl ) imide. ( A ) EIC for HMIM in a removing an ionic liquid from an aqueous sample . In some blank run (i ), after the initial ion exchange reaction ( ii ), and embodiments , the method includes : ( a ) combining an aque - two subsequent extractions ( iii and iv ) . ( B ) The same EICs ous sample including an ionic liquid with an ion exchanger as in ( A ) , with the initial ion exchange reaction EIC removed composition including an ion exchanger counterion to pro - 35 to demonstrate the levels go almost back to background . duce a solution including a fluorous salt of the ionic liquid , FIGS . 8A and 8B show EICs for bis ( (perfluorohexyl ) wherein at least one of the ionic liquid and the ion exchanger sulfonyl) imide after each extraction of the aqueous layer counterion is fluorinated ; ( b ) contacting the solution with a using fresh HFE - 7100 and bis ( (perfluorohexyl ) sulfonyl) im fluorous affinity material, thereby removing fluorous salt ide . ( A ) EICs for bis ( (perfluorohexyl ) sulfonyl) imide in a from the solution and producing an aqueous eluate ; and ( c ) 40 blank run (i ) , after the initial ion exchange reaction (ii ), and collecting the aqueous eluate . In some cases, the fluorous two subsequent extractions ( iii and iv ) . ( B ) The same EICs affinity material is an immiscible fluorous solvent that as in ( A ), with the initial ion exchange reaction EIC removed extracts the fluorous salt from the solution to produce the to demonstrate the levels go almost back to background . aqueous eluate . In certain embodiments , prior to step ( a ) , the FIG . 9 schematically illustrates a sample preparation method includes : contacting a cell with an amount of an 45 workflow for methods according one embodiment of the ionic liquid composition sufficient to lyse the cell and subject method : (2 ) Add Ionic Liquid /Water solution - Lyse produce an aqueous sample including an ionic liquid ; and Cells and quench metabolism ; ( 3 ) Entire cellular debris with contacting the aqueous sample with a reverse phase sub - Ionic Liquid /water put through C18 column to remove strate, thereby adsorbing proteins and /or lipids of the cell on proteins; (4 ) Add Aqueous Ion Exchager Solution e .g . the reverse phase substrate and producing a contacted aque - 50 LiNTf2; ( 5 ) Spin - 4000xg ; (6 ) Remove upper aqueous layer ous sample . Also provided are compositions including : a and inject in LC -MS . fluorous salt of an ionic liquid ; and a fluorous solvent. Kits FIG . 10 illustrates a photocleavable ionic liquid for use in and systems for practicing the subject methods are also methods according to an alternative embodiment of the provided . subject methods. 55 FIG . 11 schematically illustrates a workflow for methods BRIEF DESCRIPTION OF THE FIGURES according a further alternative embodiment of the subject method . IL Requirements : 1. Efficient extraction of hydro The skilled artisan will understand that the drawings , philic metabolites from water; 2 . Less dense than water ; 3 . described below , are for illustration purposes only . The After metathesis reaction , more dense than water or organic ; drawings are not intended to limit the scope of the present 60 4 . Disperses in water and organic ; 5 . Nonelutropic properties teachings in any way . by chromatographic cleanup . Note : target metabolites FIG . 1 schematically illustrates an ionic liquid metabo - include hydrophilic , polar and medium polarity. lomics sample preparation workflow for methods according FIG . 12 schematically illustrates an ionic liquid metabo one embodiment of the subject method : ( 1 ) Fast filter ; ( 2 ) lomics sample preparation workflow for methods according Lyse / Quench 50 UL 1 : 1 IL :H20 ; ( 3 ) C18 column ( remove 65 one embodiment of the subject method , using an ionic liquid protein and lipids ) ; ( 4 ) Extract IL 225 uL HO , 100 uL containing an excess of the water immiscible anion of the fluorous solvent, 1. 1 equivalents M + -NTfni (6 ) 50 °L H2O , ionic liquid (as a metal salt ) (potassium Bisnonafluoro -1 US 9 ,958 ,363 B2 butanesulfonimidate (KF -4C -NTf2 ) ) to extract the ionic cells include eukaryotic cells , such as cells obtained from liquid cation out of the aqueous solution : ( 2 ) Add Ionic biological samples from animals , plants or fungi. Liquid /Water solution -Lyse Cells and quench metabolism . The term “ denaturing ,” as used herein , refers to the ( 3 ) Entire cellular debris with lonic Liquid /water put process in which proteins or nucleic acids lose tertiary and Through C18 column to remove proteins . ( 4 ) Add Ion 5 secondary structure which is present in the native state by Exchanger Solution IL4- F4C -NTt2 + KF4C -NTt2 . ( 5 ) Spin the application of some external stress or compound , such as 4000xg ; (6 ) Remove upper aqueous layer, put onto fluorous an acid or base , a concentrated inorganic salt, an organic solvent or heat. Protein denaturation includes enzyme dena affinity column. turation where quaternary denaturation includes protein sub 10 units being dissociated or the spatial arrangement of protein DEFINITIONS subunits being disrupted . Protein denaturation may further include tertiary structure denaturation which includes the Before describing exemplary embodiments in greater disruption of covalent interactions between amino acid side detail, the following definitions are set forth to illustrate and chains ( such as disulfide bridges between cysteine groups ) , define the meaning and scope of the terms used in the 15 non - covalent dipole -dipole interactions between polar description . amino acid side chains and surrounding media , Van der Unless defined otherwise , all technical and scientific Waals interactions (e . g ., induced dipole moments ) between terms used herein have the same meaning as commonly non - polar amino acid side chains . Protein denaturation may understood by one of ordinary skill in the art to which this further include secondary structure denaturation where pro invention belongs . Singleton , et al. , DICTIONARY OF 20 teins, including enzymes lose all regular repeating patterns MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D such as alpha -helices and beta - pleated sheets and may adopt ED ., John Wiley and Sons, New York ( 1994 ), and Hale & a random - coil type configuration . Protein denaturation does Markham , THE HARPER COLLINS DICTIONARY OF not disrupt or change covalent peptide bonds or the sequence BIOLOGY, Harper Perennial, N . Y . ( 1991) provide one of of amino acids held together ( i. e ., does not disrupt primary skill with the general meaning of many of the terms used 25 structure ) . herein . Still, certain terms are defined below for the sake of The terms “ determining” , “measuring ” , “ evaluating” , clarity and ease of reference . " assessing ," " assaying, " and " analyzing ” are used inter It must be noted that as used herein and in the appended changeably herein to refer to any form of measurement, and claims, the singular forms “ a ” , “ an ” , and “ the ” include plural include determining if an element is present or not. These referents unless the context clearly dictates otherwise . For 30 terms include both quantitative and / or qualitative determi example, the term “ a primer” refers to one or more primers, nations. Assessing may be relative or absolute . “ Assessing i. e ., a single primer and multiple primers . It is further noted the presence of includes determining the amount of some that the claims can be drafted to exclude any optional thing present, as well as determining whether it is present or element. As such , this statement is intended to serve as absent . antecedent basis for use of such exclusive terminology as 35 Furthermore , except as otherwise noted , the chemical “ solely , " " only ” and the like in connection with the recita - methods and techniques of the present embodiments are tion of claim elements , or use of a " negative ” limitation . generally performed according to conventional methods The term “ sample ” as used herein relates to a material or well known in the art and as described in various general and mixture of materials, typically , although not necessarily , in more specific references that are cited and discussed liquid form , containing one or more analytes of interest. In 40 throughout the present specification . See, e . g ., Loudon , one embodiment, the term as used in its broadest sense , Organic Chemistry, Fourth Edition , New York : Oxford Uni refers to any plant, animal or bacterial material containing versity Press, 2002 , pp . 360 - 361, 1084 - 1085 ; Smith and cells or producing cellular metabolites, such as, for example , March , March 's Advanced Organic Chemistry : Reactions, tissue or fluid isolated from an individual ( including without Mechanisms, and Structure, Fifth Edition , Wiley - Inter limitation plasma , serum , cerebrospinal fluid , lymph , tears , 45 science , 2001 . saliva and tissue sections ) or from in vitro cell culture Many general references providing commonly known constituents , as well as samples from the environment. The chemical synthetic schemes and conditions useful for syn term “ sample ” may also refer to a “ biological sample” . As thesizing the disclosed compounds are available (see , e . g . , used herein , the term “ a biological sample ” refers to a whole Smith and March , March ' s Advanced Organic Chemistry : organism or a subset of its tissues, cells or component parts 50 Reactions, Mechanisms , and Structure , Fifth Edition , Wiley ( e . g . body fluids, including but not limited to blood ,mucus , Interscience , 2001; or Vogel, A Textbook of Practical lymphatic fluid , synovial fluid , cerebrospinal fluid , saliva , Organic Chemistry , Including Qualitative Organic Analysis , amniotic fluid , amniotic cord blood , urine , vaginal fluid and Fourth Edition , New York : Longman , 1978 ) . semen ) . A “ biological sample ” can also refer to a homoge Where compounds described herein contain one or more nate , lysate or extract prepared from a whole organism or a 55 chiral centers and/ or double -bond isomers ( i. e ., geometric subset of its tissues , cells or component parts , or a fraction isomers ) , enantiomers or diastereomers , all possible or portion thereof, including but not limited to , for example, enantiomers and stereoisomers of the compounds including plasma , serum , spinal fluid , lymph fluid , the external sec - the stereoisomerically pure form ( e . g . , geometrically pure , tions of the skin , respiratory, intestinal, and genitourinary enantiomerically pure or diastereomerically pure ) and tracts , tears , saliva , milk , blood cells , tumors, organs . In 60 enantiomeric and stereoisomeric mixtures are included in certain embodiments, the sample has been removed from an the description of the compounds herein . Enantiomeric and animal or plant. Biological samples of the invention include stereoisomeric mixtures can be resolved into their compo cells . The term " cells " is used in its conventional sense to nent enantiomers or stereoisomers using separation tech refer to the basic structural unit of living organisms, both niques or chiral synthesis techniques well known to the eukaryotic and prokaryotic , having at least a nucleus and a 65 skilled artisan . The compounds can also exist in several cell membrane . In certain embodiments , cells include pro - tautomeric forms including the enol form , the keto form and karyotic cells , such as from bacteria . In other embodiments, mixtures thereof . Accordingly , the chemical structures US 9 , 958 , 363 B2 depicted herein encompass all possible tautomeric forms of cycloalkyl , cycloheteroalkyl, aryl , arylalkyl, heteroalkyl, the illustrated compounds . The compounds described also heteroaryl , heteroarylalkyl as defined herein and substituted include isotopically labeled compounds where one or more versions thereof. Representative examples include, but are atoms have an atomic mass different from the atomic mass not limited to formyl, acetyl, cyclohexylcarbonyl , cyclohex conventionally found in nature . Examples of isotopes that 5 ylmethylcarbonyl, benzoyl, benzylcarbonyl, piperonyl, suc can be incorporated into the compounds disclosed herein cinyl, and malonyl, and the like . include , but are not limited to , 2H , PH , 110 , 14C , 15N , 180 , The term “ aminoacyl ” refers to the group - C ( O ) 170 , etc . Compounds can exist in unsolvated forms as well NR2R22, wherein R21 and R22 independently are selected as solvated forms, including hydrated forms. In general, from the group consisting of hydrogen , alkyl, substituted compounds can be hydrated or solvated . Certain compounds 10 alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alky can exist in multiple crystalline or amorphous forms. In nyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl , general , all physical forms are equivalent for the uses cycloalkenyl, substituted cycloalkenyl, heteroaryl, substi contemplated herein and are intended to be within the scope tuted heteroaryl, heterocyclic , and substituted heterocyclic of the present disclosure . and where R21 and R22 are optionally joined together with As used herein , the term “ alkyl” by itself or as part of 15 the nitrogen bound thereto to form a heterocyclic or substi another substituent refers to a saturated branched or straight- tuted heterocyclic group , and wherein alkyl, substituted chain monovalent hydrocarbon radical derived by the alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alky removal of one hydrogen atom from a single carbon atom of nyl , cycloalkyl , substituted cycloalkyl, cycloalkenyl, substi a parent alkane . Typical alkyl groups include , but are not tuted cycloalkenyl , aryl, substituted aryl, heteroaryl , substi limited to , methyl; ethyl, propyls such as propan - 1- yl or 20 tuted heteroaryl , heterocyclic , and substituted heterocyclic propan - 2 - yl; and butyls such as butan - 1 - yl, butan - 2 - yl, are as defined herein . 2 -methyl - propan - 1 - yl or 2 -methyl - propan - 2 - yl. In some “ Alkoxy ” by itself or as part of another substituent refers embodiments , an alkyl group includes from 1 to 20 carbon to a radical OR31 where R31 represents an alkyl or atoms. In other embodiments , an alkyl group includes from cycloalkyl group as defined herein . Representative examples 1 to 10 carbon atoms. In still other embodiments , an alkyl 25 include , but are not limited to , methoxy , ethoxy , propoxy , group includes from 1 to 6 carbon atoms, such as from 1 to butoxy, cyclohexyloxy and the like . 4 carbon atoms. “ Alkoxycarbonyl” by itself or as part of another substitu " Alkanyl ” by itself or as part of another substituent refers ent refers to a radical - C ( O )OR31 where R31 represents an to a saturated branched , straight - chain or cyclic alkyl radical alkyl or cycloalkyl group as defined herein . Representative derived by the removal of one hydrogen atom from a single 30 examples include , but are not limited to , methoxycarbonyl, carbon atom of an alkane. Typical alkanyl groups include , ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, cyclo but are not limited to , methanyl; ethanyl; propanyls such as hexyloxycarbonyl and the like. propan - 1 - yl , propan - 2 - yl (isopropyl ) , cyclopropan - 1 - yl, “ Aryl ” by itself or as part of another substituent refers to etc . ; butanyls such as butan - 1 - yl, butan - 2 -yl ( sec -butyl ) , a monovalent aromatic hydrocarbon radical derived by the 2 -methyl - propan - 1 - yl ( isobutyl) , 2 -methyl - propan - 2 -yl 35 removal of one hydrogen atom from a single carbon atom of (t - butyl) , cyclobutan - 1 - yl, etc .; and the like . an aromatic ring system . Typical aryl groups include, but are " Alkylene” refers to a branched or unbranched saturated not limited to , groups derived from aceanthrylene , acenaph hydrocarbon chain , usually having from 1 to 40 carbon thylene , acephenanthrylene, anthracene, azulene , benzene , atoms, more usually 1 to 10 carbon atoms and even more chrysene , coronene , fluoranthene, fluorene , hexacene , hexa usually 1 to 6 carbon atoms. This term is exemplified by 40 phene, hexalene , as- indacene, s -indacene , indane, indene , groups such as methylene ( CH2 – ) , ethylene naphthalene, octacene, octaphene , octalene, ovalene , penta CH , CH , - ) , the propylene isomers ( e . g ., 2 , 4 -diene , pentacene , pentalene , pentaphene , perylene , phe — CH ,CH , CH , - and - CH (CH3 ) CH , - ) and the like . nalene , phenanthrene , picene , pleiadene , pyrene , pyran “ Alkenyl ” by itself or as part of another substituent refers threne , rubicene , triphenylene, trinaphthalene and the like . to an unsaturated branched , straight -chain or cyclic alkyl 45 In certain embodiments , an aryl group includes from 6 to 20 radical having at least one carbon -carbon double bond carbon atoms. In certain embodiments , an aryl group derived by the removal of one hydrogen atom from a single includes from 6 to 12 carbon atoms. Examples of an aryl carbon atom of an alkene . The group may be in either the cis group are phenyl and naphthyl. or trans conformation about the double bond ( s ) . Typical “ Arylalkyl” by itself or as part of another substituent alkenyl groups include, but are not limited to , ethenyl; 50 refers to an acyclic alkyl radical in which one of the propenyls such as prop - 1- en - 1 -yl , prop - 1- en - 2 -yl , prop - 2 hydrogen atoms bonded to a carbon atom , typically a en - 1 - yl ( allyl ) , prop - 2 - en - 2 - yl, cycloprop - 1 - en - 1 - yl ; cyclo - terminal or sp carbon atom , is replaced with an aryl group . prop - 2 - en - 1 -yl ; butenyls such as but - 1 -en - 1 -yl , but- 1 - en - 2 - Typical arylalkyl groups include , but are not limited to , yl, 2 -methyl - prop - 1 -en - 1 -yl , but- 2 -en - 1 -yl , but- 2 -en - 1- yl, benzyl, 2 -phenylethan - 1- yl, 2 - phenylethen - 1 -yl , naphthylm but- 2 - en - 2 - yl, buta - 1 , 3 - dien - 1 - yl, buta - 1 , 3 - dien - 2 - yl, 55 ethyl, 2 -naphthylethan - 1 - yl, 2 - naphthylethen - 1 - yl, naph cyclobut- 1 - en - 1 - yl, cyclobut- 1 - en - 3 - yl, cyclobuta - 1 , 3 - dien thobenzyl, 2 - naphthophenylethan - 1 - yl and the like . Where 1 - yl, etc . ; and the like . specific alkyl moieties are intended , the nomenclature ary " Alkynyl” by itself or as part of another substituentrefers lalkanyl, arylalkenyl and / or arylalkynyl is used . In certain to an unsaturated branched , straight - chain or cyclic alkyl embodiments , an arylalkyl group is (C7 - C30 ) arylalkyl, e . g ., radical having at least one carbon - carbon triple bond derived 60 the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group by the removal of one hydrogen atom from a single carbon is (C1 - C10 ) and the aryl moiety is (Co - C20 ) . In certain atom of an alkyne. Typical alkynyl groups include , but are embodiments , an arylalkyl group is ( Cz- C20 ) arylalkyl, e . g . , not limited to , ethynyl; propynyls such as prop - 1- yn - 1- yl , the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group prop -2 -yn -1 -yl , etc .; butynyls such as but- 1 -yn - 1 -yl , but- 1 - is (C , - C ) and the aryl moiety is (Co - C12 ). yn - 3 -yl , but- 3 - yn - 1 -yl , etc .; and the like . 65 “ Arylaryl” by itself or as part of another substituent, refers “ Acyl” by itself or as part of another substituent refers to to a monovalent hydrocarbon group derived by the removal a radical _ C (O )R30 , where R30 is hydrogen , alkyl, of one hydrogen atom from a single carbon atom of a ring US 9 ,958 ,363 B2 system in which two or more identical or non - identical oxazole , perimidine , phenanthridine, phenanthroline , aromatic ring systemsare joined directly together by a single phenazine , phthalazine , pteridine , purine, pyran , pyrazine , bond , where the number of such direct ring junctions is one pyrazole , pyridazine, pyridine, pyrimidine, pyrrole , pyr less than the number of aromatic ring systems involved . rolizine, quinazoline, quinoline , quinolizine , quinoxaline , Typical arylaryl groups include , but are not limited to , 5 tetrazole , thiadiazole , thiazole , thiophene , triazole , xan biphenyl, triphenyl, phenyl- napthyl, binaphthyl , biphenyl- thene , benzodioxole and the like . In certain embodiments , napthyl, and the like. When the number of carbon atoms in the heteroaryl group is from 5 - 20 membered heteroaryl. In an arylaryl group are specified , the numbers refer to the certain embodiments , the heteroaryl group is from 5 - 10 carbon atoms including each aromatic ring . For example , membered heteroaryl. In certain embodiments , heteroaryl (C3 - C14 ) arylaryl is an arylaryl group in which each aro - 10 groups are those derived from thiophene , pyrrole , benzoth matic ring includes from 5 to 14 carbons , e . g ., biphenyl, iophene , benzofuran , indole , pyridine , quinoline , imidazole , triphenyl, binaphthyl, phenylnapthyl, etc . In certain embodi oxazole and pyrazine . ments, each aromatic ring system of an arylaryl group is “ Heteroarylalkyl” by itself or as part of another substitu independently a (C3 - C14 ) aromatic . In certain embodiments , ent, refers to an acyclic alkyl radical in which one of the each aromatic ring system of an arylaryl group is indepen - 15 hydrogen atoms bonded to a carbon atom , typically a dently a (CG - C ) aromatic . In certain embodiments , each terminal or spa carbon atom , is replaced with a heteroaryl aromatic ring system is identical, e . g ., biphenyl, triphenyl, group . Where specific alkyl moieties are intended , the binaphthyl, trinaphthyl, etc . nomenclature heteroarylalkanyl, heteroarylalkenyl and /or " Cycloalkyl” by itself or as part of another substituent heterorylalkynyl is used . In certain embodiments , the het refers to a saturated or unsaturated cyclic alkyl radical. 20 eroarylalkyl group is a 6 - 30 membered heteroarylalkyl, e. g ., Where a specific level of saturation is intended , the nomen - the alkanyl , alkenyl or alkynyl moiety of the heteroarylalkyl clature " cycloalkanyl" or " cycloalkenyl” is used . Typical is 1 - 10 membered and the heteroaryl moiety is a 5 - 20 cycloalkyl groups include , but are not limited to , groups membered heteroaryl. In certain embodiments , the het derived from cyclopropane , cyclobutane , cyclopentane , eroarylalkyl group is 6 - 20 membered heteroarylalkyl, e . g . , cyclohexane and the like . In certain embodiments , the 25 the alkanyl, alkenyl or alkynylmoiety of the heteroarylalkyl cycloalkyl group is (Cz - C10 ) cycloalkyl. In certain embodi- is 1 - 8 membered and the heteroaryl moiety is a 5 - 12 ments, the cycloalkyl group is (C3 - C7) cycloalkyl . membered heteroaryl. “ Cycloheteroalkyl” or “ heterocyclyl” by itself or as part “ Aromatic Ring System ” by itself or as part of another of another substituent, refers to a saturated or unsaturated substituent, refers to an unsaturated cyclic or polycyclic ring cyclic alkyl radical in which one or more carbon atoms (and 30 system having a conjugated at electron system . Specifically any associated hydrogen atoms) are independently replaced included within the definition of “ aromatic ring system ” are with the same or different heteroatom . Typical heteroatoms fused ring systems in which one or more of the rings are to replace the carbon atom ( s ) include , but are not limited to , aromatic and one or more of the rings are saturated or N , P , O , S , Si, etc . Where a specific level of saturation is unsaturated , such as, for example , fluorene, indane , indene , intended , the nomenclature " cycloheteroalkanyl" or " cyclo - 35 phenalene , etc . Typical aromatic ring systems include , but heteroalkenyl” is used . Typical cycloheteroalkyl groups are not limited to , aceanthrylene , acenaphthylene , ace include , but are not limited to , groups derived from epox - phenanthrylene , anthracene , azulene , benzene , chrysene , ides , azirines , thiiranes , imidazolidine , morpholine , pipera - coronene , fluoranthene , fluorene, hexacene , hexaphene , zine, piperidine, pyrazolidine, pyrrolidine , quinuclidine and hexalene , as - indacene , s - indacene , indane, indene , naphtha the like . 40 lene , octacene , octaphene , octalene , ovalene , penta - 2 , 4 - di “ Heteroalkyl, Heteroalkanyl, Heteroalkenyl and Het - ene , pentacene, pentalene, pentaphene, perylene , phenalene , eroalkynyl ” by themselves or as part of another substituent phenanthrene , picene, pleiadene , pyrene , pyranthrene , rubi refer to alkyl, alkanyl, alkenyl and alkynyl groups , respec cene, triphenylene , trinaphthalene and the like . tively, in which one or more of the carbon atoms ( and any “ Heteroaromatic Ring System ” by itself or as part of associated hydrogen atoms) are independently replaced with 45 another substituent, refers to an aromatic ring system in the same or different heteroatomic groups . Typical hetero - which one or more carbon atoms ( and any associated atomic groups which can be included in these groups hydrogen atoms) are independently replaced with the same include, but are not limited to , - o - , - S — , - S - S — or different heteroatom . Typical heteroatoms to replace the -O S NR37R38 = N / N = , — N — N — carbon atoms include, but are not limited to , N , P , O , S , Si, - N = N — NR39R40 , PR41 % , P ( O ) 2 - , - POR42 — , 50 etc . Specifically included within the definition of " heteroaro - 0 — P ( 0 )2 - , - S40 - , S - 0 ) , S02 — , matic ring systems” are fused ring systems in which one or - SnR43R44 — and the like , where R37 , R38 , R39 , R40 , R41, more of the rings are aromatic and one or more of the rings R42, R43 , and R44 are independently hydrogen , alkyl, sub are saturated or unsaturated , such as, for example , arsindole, stituted alkyl, aryl, substituted aryl, arylalkyl, substituted benzodioxan , benzofuran , chromane , chromene , indole , arylalkyl, cycloalkyl, substituted cycloalkyl , cyclohet - 55 indoline, xanthene , etc . Typical heteroaromatic ring systems eroalkyl, substituted cycloheteroalkyl, heteroalkyl, substi - include, but are not limited to , arsindole , carbazole , ß -car tuted heteroalkyl, heteroaryl, substituted heteroaryl, het boline , chromane , chromene, cinnoline , furan , imidazole , eroarylalkyl or substituted heteroarylalkyl. indazole , indole , indoline , indolizine , isobenzofuran , iso “ Heteroaryl” by itself or as part of another substituent, chromene , isoindole , isoindoline , isoquinoline , isothiazole , refers to a monovalent heteroaromatic radical derived by the 60 isoxazole , naphthyridine, oxadiazole , oxazole , perimidine , removal of one hydrogen atom from a single atom of a phenanthridine , phenanthroline , phenazine , phthalazine , heteroaromatic ring system . Typical heteroaryl groups pteridine , purine , pyran , pyrazine , pyrazole , pyridazine , include , but are not limited to , groups derived from acridine , pyridine, pyrimidine , pyrrole , pyrrolizine , quinazoline , qui arsindole, carbazole , ß -carboline , chromane , chromene , cin - noline , quinolizine , quinoxaline , tetrazole, thiadiazole , thi noline , furan , imidazole , indazole , indole , indoline , indoliz - 65 azole , thiophene , triazole , xanthene and the like. ine , isobenzofuran , isochromene , isoindole , isoindoline, iso “ Substituted ” refers to a group in which one or more quinoline , isothiazole, isoxazole , naphthyridine, oxadiazole , hydrogen atoms are independently replaced with the same or US 9 ,958 ,363 B2 10 different substituent( s ) . Typical substituents include, but are The term “ analyte ” are used herein interchangeably and not limited to , alkylenedioxy ( such asmethylenedioxy ), -M , refer to a known or unknown component of a sample , which - R60, 0 , = O , OR , SR , S , = S , will specifically bind to a capture agent on a substrate - NR “ OR?I , = NR “ , CF3 , CN , OCN , SCN , surface if the analyte and themolecular probe are members 5 of a specific binding pair . In general , analytes are biopoly =- N2NO, , - N3NO2, - S ( O )20 - , - S ( O ) , OH , - S ( O ) , R “ , OS mers , i . e ., an oligomer or polymer such as an oligonucle (0 )20 " , OS( O ), R , P (O )( 0 ) 2, P ( O )( OR )( 0 - ), otide, a peptide , a polypeptide, an antibody, or the like . In - OP ( O ) ( OR60 ) ( OR61 ) , C ( O ) R60 , - C ( S ) R 60, CTO ) this case , an “ analyte ” is referenced as a moiety in a mobile OR60 , - C ( O )NRR61 , - C ( O ) O - , - C ( S )OR60 , - NR62C phase ( typically fluid ) , to be detected by a “ capture agent” ( O )NRR61 , - NR62C ( S ) NRR61 , - NR62C (NR63 ) 10 which , in most embodiments , is bound to a substrate . NRR61 and C (NR62 ) NR6R61 where M is halogen ; RC , However , either of the “ analyte ” or “ capture agent” may be R61 , R62 and R63 are independently hydrogen , alkyl, substi the one which is to be evaluated by the other ( thus , either one tuted alkyl , alkoxy, substituted alkoxy , cycloalkyl, substi could be an unknown mixture of analytes, e .g ., polypeptides , tuted cycloalkyl, cycloheteroalkyl, substituted cyclohet - coulto be evaluated by binding with the other ). eroalkyl, aryl , substituted aryl, heteroaryl or substituted 15 The term “ biological sample ” is used herein to refer to a heteroaryl, or optionally Rº and Rº together with the whole organism , plant, fungi or a subset of animal tissues , nitrogen atom to which they are bonded form a cyclohet - cells or component parts which may in certain instances be eroalkyl or substituted cycloheteroalkyl ring ; and Rº4 and found in blood , mucus, lymphatic fluid , synovial fluid , R65 are independently hydrogen , alkyl, substituted alkyl, cerebrospinal fluid , saliva , amniotic fluid , amniotic cord aryl, cycloalkyl , substituted cycloalkyl, cycloheteroalkyl, w blood , urine , vaginal fluid and semen . As such , a “ biological substituted cycloheteroalkyl, aryl, substituted aryl, het- 4 sample ” used herein can refer to a homogenate, lysate or eroaryl or substituted heteroaryl, or optionally R64 and RÓS extract prepared from a whole organism or a subset of its together with the nitrogen atom to which they are bonded tissues, including but not limited to , for example , plasma, form a cycloheteroalkyl or substituted cycloheteroalkyl ring . serum , spinal fluid , lymph fluid , the external sections of the In certain embodiments , substituents include - M , RO skin , respiratory , intestinal, and genitourinary tracts , tears , — O , OR60 , - SR60 , - S - , GS , NR R1, = NR , 25 saliva , milk , blood cells , tumors , organs . In embodiments of - CF3, CN , OCN , SCN , NO , NO , the invention , a “ biological sample ” will contain cells from = N , , - N2, - S ( O ) , R “ , - OS ( O ) , 0 - , - OS ( O ) , Rº , P ( O ) the animal, plants , bacteria or fungi. A “ biological sample " (0 )2 , - P (O ) (OR 0 (0 -) , OP (O )( OR ) (ORT ), CO ) can also refer to a medium , such as a nutrient broth or gel R “ , - C ( S )RÓ , COJOR , CONRR61, - C ( O ) O - , in which an organism has been propagated , which contains - NR62C ( O )NRR61 . In certain embodiments , substituents 30 cells as well as cellular components , such as proteins or include - M , RO, nucleic acid molecules. Biological samples of the invention O , OR “ , SR , NR R1, CF3, CN , NOZ, include cells . The term " cells ” is used in its conventional - S (O ), R " , OP ( O ) (OR ) (ORI ), - C (O ) R “ , - C ( O ) sense to refer to the basic structural unit of living organisms, ORO, CONRRO , C 0 ) O . In certain embodiments , both eukaryotic and prokaryotic , having at least a nucleus substituents include - M , RO, 35 and a cell membrane . In certain embodiments , cells include = O , OR , SR , — NRØRT, CF3, CN , — NO , , prokaryotic cells , such as from bacteria . In other embodi - S ( O ), R “ , - C ( O ) R “ , - C ( O ) OR “ , - C ( O ) O - , where ments , cells include eukaryotic cells , such as cells obtained R6 , R1 and R?2 are as defined above . For example , a from biological samples from animals , plants or fungi. substituted group may bear a methylenedioxy substituent or The methods described herein include multiple steps . one, two , or three substituents selected from a halogen atom , Each step may be performed after a predetermined amount a ( 1 - 4C ) alkyl group and a ( 1 - 4C ) alkoxy group . of time has elapsed between steps, as desired . As such , the A plurality ” contains at least 2 members . In certain cases , time between performing each step may be 1 second or a plurality may have at least 10 , at least 100 , at least 100 , at more , 10 seconds or more , 30 seconds or more , 60 seconds least 10 ,000 , at least 100 ,000 , at least 10 % , at least 10 % , at or more , 5 minutes or more , 10 minutes or more , 60 minutes least 108 or at least 10° or more members . or more and including 5 hours or more . In certain embodi Numeric ranges are inclusive of the numbers defining the 45 ments , each subsequent step is performed immediately after range. completion of the previous step . In other embodiments , a The term " separating ” , as used herein , refers to physical step may be performed after an incubation or waiting time separation of two elements ( e . g ., by size or affinity , etc . ) as after completion of the previous step , e . g . , a few minutes to well as degradation of one element, leaving the other intact. an overnight waiting time. The term “ sample ” as used herein relates to a material or 50 Other definitions of terms may appear throughout the mixture of materials , typically , although not necessarily , in specification . fluid , i. e ., aqueous , form , containing one or more compo nents of interest. Samples may be derived from a variety of DESCRIPTION OF EXEMPLARY sources such as from food stuffs, environmental materials , a EMBODIMENTS biological sample or solid , such as tissue or fluid isolated from an individual, including but not limited to , for example , As summarized above, aspects of the present disclosure plasma, serum , spinal fluid , semen , lymph fluid , the external include a method for removing an ionic liquid from an sections of the skin , respiratory, intestinal , and genitourinary aqueous sample . In some embodiments , the method tracts , tears, saliva , milk , blood cells , tumors, organs, and includes : ( a ) combining an aqueous sample including an also samples of in vitro cell culture constituents ( including ionic liquid with an ion exchanger composition including an but not limited to conditioned medium resulting from the 60 ion exchanger counterion to produce a solution including a growth of cells in cell culture medium , putatively virally fluorous salt of the ionic liquid , wherein at least one of the infected cells , recombinant cells , and cell components ) . ionic liquid and the ion exchanger counterion is fluorinated ; Components in a sample may be termed " analytes ” . In ( b ) contacting the solution with a fluorous affinity material, many embodiments , the sample is a complex sample con thereby removing fluorous salt from the solution and pro taining at least about 102, 5x102, 103, 5x103, 104, 5x104, 65 ducing an aqueous eluate ; and ( c ) collecting the aqueous 10 %, 5x105 , 106, 5x106, 107, 5x107, 108, 109, 1010 , 1011 eluate . In some cases , the fluorous affinity material is an 1012 or more species of analyte . immiscible fluorous solvent that extracts the fluorous salt US 9 ,958 ,363 B2 12 from the solution to produce the aqueous eluate . In certain aqueous sample includes an analyte of interest , such as a embodiments , prior to step ( a ), the method includes : con metabolite of interest ( e . g ., as described herein ) . The subject tacting a cell with an amount of an ionic liquid composition methods find use in removing an ionic liquid from an sufficient to lyse the cell and produce an aqueous sample aqueous sample to provide a sample suitable for analysis of including an ionic liquid ; and contacting the aqueous sample 5 a target analyte . In some cases , the ionic liquid has a with a reverse phase substrate , thereby adsorbing proteins detrimental effect on the analysis of the aqueous sample and and/ or lipids of the cell on the reverse phase substrate and removal of the ionic liquid provides for the elimination of producing a contacted aqueous sample . Also provided are this detrimental effect. In some cases , the aqueous sample is compositions including : a fluorous salt of an ionic liquid ; derived from a biological sample that includes an ionic and a fluorous solvent . Kits and systems for practicing the 10 liquid such as a cellular sample ( e . g . , as described herein ) . subject methods are also provided . In certain instances, the target analyte is a metabolite . In Before the various embodiments are described , it is to be certain instances, the target analyte is a protein . In certain understood that the teachings of this disclosure are not instances , the target analyte is a lipid . limited to the particular embodiments described , and as such The subject methods and compositions find use in the can , of course , vary . It is also to be understood that the 15 removal of an ionic liquid from an aqueous sample of terminology used herein is for the purpose of describing interest using any one of a variety ofmethods including, but particular embodiments only , and is not intended to be not limited to , extraction , chromatography , immobilization limiting , since the scope of the present teachings will be and separation ( e . g . , using a magnetic field ) . In general limited only by the appended claims. terms, the method includes producing a salt of the ionic The section headings used herein are for organizational liquid and an ion exchanger counterion , which salt may have purposes only and are not to be construed as limiting the - a physical or chemical property that provides for preferential subject matter described in any way. While the present removal of the salt from the aqueous sample using a suitable teachings are described in conjunction with various embodi method ( e . g . , as described herein ) . In some instances , an ments , it is not intended that the present teachings be limited ionic liquid may be removed via the immobilization of a to such embodiments . On the contrary , the present teachings magnetic composition , e . g ., via the application of a magnetic encompass various alternatives, modifications, and equiva - 25 field to isolate a magnetic salt of the ionic liquid and the ion lents , as will be appreciated by those of skill in the art . exchanger. In some cases , an ionic liquid may be removed Unless defined otherwise , all technical and scientific using click chemistry , e . g . , where the salt of the ionic liquid , terms used herein have the same meaning as commonly or the ionic liquid itself is engineered to include a chemose understood by one of ordinary skill in the art to which this lective tag that provides for immobilization or separation disclosure belongs . Although any methods and materials 30 from the aqueous sample . In some cases , an ionic liquid may similar or equivalent to those described herein can also be be removed using a photocleavable group , e . g ., via removal used in the practice or testing of the present teachings , some of a photocleavable ionic liquid or salt thereof from a sample exemplary methods and materials are now described . by application of light to photocleave the ionic liquid . The citation of any publication is for its disclosure prior Cleavage of the ionic liquid may provide two or more to the filing date and should not be construed as an admis - 35 fragments , where some of the fragments produced are more sion that the present claims are not entitled to antedate such easily extracted or separated from the sample of interest . In publication by virtue of prior invention . Further , the dates of certain embodiments , ionic liquids may be removed from an publication provided can be different from the actual pub - aqueous sample via fluorous affinity interactions of a fluo lication dates which can be independently confirmed . rorus salt of the ionic liquid and an ion exchanger with a As will be apparent to those of skill in the art upon reading fluorous affinity substrate . In certain instances, the fluorous this disclosure , each of the individual embodiments tu salt is removed using fluorous affinity chromatography . In described and illustrated herein has discrete components and certain instances , the fluorous salt is removed via extraction features which can be readily separated from or combined with a fluorous liquid . with the features of any of the other several embodiments Ionic Liquids without departing from the scope or spirit of the present An ionic liquid is a salt in which counterions are poorly teachings. Any recited method can be carried out in the order 45 coordinated , and which results in the salts being in liquid of events recited or in any other order which is logically form below 100° C . The term “ ionic liquid ” is used in its possible . conventional sense to refer to a salt in liquid state . Ionic All patents and publications, including all sequences liquids of interest are compounds in the liquid state at room disclosed within such patents and publications , referred to temperature that are made of ions or short - lived ion pairs and herein are expressly incorporated by reference . 50 may alternatively be referred to as liquid electrolytes, ionic In further describing the subject invention , methods and melts , ionic fluids , fused salts , liquid salts or ionic glasses . compositions for removing an ionic liquid from an aqueous As such , ionic liquids may include salts composed of ion sample , are described . Next, methods and compositions for pairs that are in the liquid state at room temperature . In some extracting and purifying compounds from a biological cases, at least one ion in the ionic salt has a delocalized sample having cells using an ionic liquid are reviewed . ss charge . In some cases, at least one ion in the ionic salt is Compositions , kits and systems are also described . organic , which prevents the formation of a stable crystal Methods for Removing an Ionic Liquid from an Aqueous lattice . In some embodiments , the ionic salt includes an Sample organic cation and an inorganic anion . In certain embodi Aspects of the present disclosure include a method for ments , the inorganic anion is a chloride . In some embodi removing an ionic liquid from an aqueous sample . Ionic ments, the ionic salt includes an organic anion and an liquids are a class of diverse organic salts having relatively 60 inorganic cation . In certain embodiments , the inorganic low melting points which find use in a variety of applica cation is a monovalent metal ion , such as a sodium , potas tions , e . g. , as organic solvent substitutes, or cellmembrane sium or lithium . Any convenient ionic salts may find use in disrupters . the subject methods and compositions . Ionic salts of interest Any convenient aqueous samples that include an ionic salt include , but are not limited to , imidazolium salts , pyridinium may find use in the subject methods . Samples of interest 65 salts , pyrrolidinium salts, phosphonium salts, ammonium include , but are not limited to , food samples, environmental salts, sulfonium salts , alkylsulfate salts , tosylate salts , meth samples and biological samples . In some embodiments , the anesulfonate salts , bis ( trifluoromethyl- sulfonyl) imide salts , US 9 ,958 ,363 B2 13 14 hexafluoro - phosphate salts , and tetrafluoro - borate salts . In In some embodiments , the ionic liquid includes a cation some embodiments , the ionic liquid includes a cation of Formula ( V ) : selected from the group consisting of sulfonium cations , phosphonium cations , tetraalkyl ammonium cations and pyrazolium cations . In certain embodiments , the ionic liquid 5 is hydrophilic . In some embodiments, the ionic liquid is hydrophobic . R - NO In some embodiments , the ionic liquid includes a cation of Formula ( I ) : 10 where R is hydrogen , alkyl, substituted alkyl, aryl, substi tuted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, sub (1 ) stituted heteroalkyl , heteroaryl, substituted heteroaryl, het eroarylalkyl or substituted heteroarylalkyl. R N- N R2 In some embodiments , the ionic liquid includes a cation 15 of Formula (VI ) : where each of Rl and R² is independently hydrogen , alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted ( VI) arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted het - 20 RO ) eroarylalkyl. R21 In some embodiments , the ionic liquid includes a cation of Formula (II ): where each of Rl and R2 is independently hydrogen, alkyl , os substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl , substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted het eroarylalkyl. In certain embodiments , the ionic liquid includes a cation 30 having Formula ( I) : Z-A

where R is hydrogen , alkyl, substituted alkyl, aryl, sub RI N- N - R2 stituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, 35 substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl. where each of R ' and R2 is independently hydrogen, alkyl , In some embodiments , the ionic liquid includes a cation substituted alkyl, aryl, substituted aryl, arylalkyl, substituted of Formula (111 ) : arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, 40 substituted heteroaryl, heteroarylalkyl, substituted het eroarylalkyl. In some instances of formula (I ), R ' is alkyl or ( 111 ) a substituted alkyl and R² is alkyl or a substituted alkyl. In some instances of formula ( I ) , R ' and R are independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, 45 heptyl and octyl. In some instances of formula ( I) , R ' is R methyl and R2 is butyl. Ionic liquids of interest include , but are not limited to where R is hydrogen , alkyl, substituted alkyl, aryl, substi 1 -butyl - 3 -methyl - imidazol- 3 - ium . In certain embodiments , tuted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, sub the ionic liquid is a compound selected from the group stituted heteroalkyl , heteroaryl, substituted heteroaryl, het- 50 consisting of 1, 2 ,4 - trimethylpyrazolium methylsulfate , eroarylalkyl or substituted heteroarylalkyl. methyl- trioctylammonium bis ( trifluoromethylsulfonyl) In some embodiments , the ionic liquid includes a cation imide, trihexyltetradecylphosphonium bromide and 5 - ( trif of Formula ( IV ) : luoromethyl) dibenzothiophenium trifluoromethanesul 55 fonate. In some embodiments , the ionic liquid includes ( IV ) 1 - hexyl - 3 -methyl - imidazolium . In some embodiments, the ionic liquid includes an anion A-Z ( e . g ., an organic anion ) , such as an anion selected from the group consisting of a carboxylate , a phosphate ester , a + sulfonate , and a borate . Any convenient anions may be utilized in the subject ionic liquids . In certain embodiments , the ionic liquid is fluorinated . In some instances, the ionic liquid includes a perfluorinated where R is hydrogen , alkyl, substituted alkyl, aryl, substi group . Any convenient fluorinated derivatives of ionic liq tuted aryl, arylalkyl , substituted arylalkyl , heteroalkyl, sub - 65 uids may be utilized . In certain embodiments , the fluorinated stituted heteroalkyl, heteroaryl, substituted heteroaryl, het- ionic liquid includes a cation selected from the group eroarylalkyl or substituted heteroarylalkyl. consisting of: US 9 , 958 , 363 B2 15 16 a ) Formula ( I ) : where each of R ' and R² is independently hydrogen , alkyl, perfluoroalkyl, a substituted alkyl, aryl, perfluoroaryl, sub stituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, perfluoro substituted het R - NON - R ?, 5 eroaryl, heteroarylalkyl, substituted heteroarylalkyl, or fluo rinated or perfluorinated derivatives thereof; or f) Formula (VI ): where each ofRand R² is independently hydrogen , alkyl, perfluoroalkyl , a substituted alkyl, aryl, perfluoroaryl, sub

stituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, 10 ? substituted heteroalkyl, heteroaryl , perfluoro substituted het eroaryl, heteroarylalkyl, substituted heteroarylalkyl, or fluo R2 rinated or perfluorinated derivatives thereof; or b ) Formula (II ): where each of R ' and R2 is independently hydrogen , alkyl , perfluoroalkyl, a substituted alkyl, aryl, perfluoroaryl, sub stituted aryl , arylalkyl, substituted arylalkyl, heteroalkyl , substituted heteroalkyl, heteroaryl, perfluoro substituted het eroaryl, heteroarylalkyl, substituted heteroarylalkyl, or fluo rinated or perfluorinated derivatives thereof. In certain embodiments , the fluorinated ionic liquid OZ-A includes a cation having Formula ( I ) : where R is hydrogen , alkyl, perfluoroalkyl, a substituted alkyl, aryl, perfluoroaryl, substituted aryl, arylalkyl, substi - 25 tuted arylalkyl, heteroalkyl , substituted heteroalkyl , het R - NON - R2 eroaryl, perfluoro substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, or fluorinated or perfluorinated where each of R ' and R2 is independently hydrogen , alkyl , derivatives thereof; or 30 perfluoroalkyl, a substituted alkyl, aryl, perfluoroaryl , sub c) Formula ( III) : stituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, perfluoro substituted het eroaryl, heteroarylalkyl, substituted heteroarylalkyl, or fluo rinated or perfluorinated derivatives thereof. 35 In certain embodiments , the ionic liquid includes a cation OR having Formula ( I ): where R is hydrogen , alkyl, perfluoroalkyl, a substituted alkyl, aryl, perfluoroaryl, substituted aryl, arylalkyl, substi tuted arylalkyl, heteroalkyl, substituted heteroalkyl, het - 4 R1 N- N - R2 eroaryl, perfluoro substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, or fluorinated or perfluorinated where each of Rl and R² is independently an alkyl, a derivatives thereof; or substituted alkyl (such as a fluorinated alkyl) . In certain d ) Formula ( IV ) : 45 instances, R and R2 are each independently a perfluorinated alkyl. In some instances , the ionic liquid includes a fluorinated A-Z derivative of any one of the cations of Formulae ( I ) to (VI ) , e . g . , as described above . In certain embodiments , fluorinated 50 ionic liquids of interest include 1 -butyl - 3 -methyl - imidazol + 3 - ium , wherein the 1 - butyl and 3 - methyl substituents are fluorinated . Reverse Phase Chromatography where R is hydrogen , alkyl , perfluoroalkyl, a substituted Aspects of the method include the use of reverse phase alkyl, arvl, perfluoroarvl. substituted arvl. arvialkyl, substi - 55 chromatography to adsorb proteins and / or lipids that may be present in the aqueous sample . In some embodiments, the tuted arylalkyl, heteroalkyl, substituted heteroalkyl, het method further includes , contacting the aqueous sample eroaryl, perfluoro substituted heteroaryl, heteroarylalkyl, including an ionic liquid with a reverse phase substrate , substituted heteroarylalkyl, or fluorinated or perfluorinated thereby adsorbing proteins and / or lipids on the reverse phase derivatives thereof; or 60 substrate , if present in the aqueous sample . In some e ) Formula ( V ) : instances, the aqueous sample is a cellular sample that includes an ionic liquid . As such , the cellular sample may be one to which an ionic liquid has been added to lyse the cells and / or quench metabolism of the cells in the sample ( e . g . , as R - NO 65 described herein ) . In such cases, removal of proteins and / or lipids from the sample ensures metabolism remains quenched , e .g ., by removing metabolic enzymes from ana US 9 ,958 ,363 B2 18 lytes of interest . In some embodiments, the method further Any convenient fluorinated ( e . g . , perfluorinated ) counte includes lysing cells of a biological sample; and contacting rions may be utilized in the subject ion exchange composi a biological sample with an amount of the ionic liquid tions. As used herein , the term " fluorinated counterion ” sufficient to denature intracellular metabolic enzymes in the refers to a counterion of the ion exchanger composition biological sample to produce the aqueous sample . The 5 capable of forming a salt with an ionic liquid of interest , proteins and / or lipids of interest from the sample that are where the counterion is fluorinated . As such , the term adsorbed on a reverse phase chromatography support and fluorinated when used herein in the context of a “ fluorinated separated from the aqueous sample may be subsequently counterion ” refers to both fluorinated and perfluorinated eluted from the support . In such cases , any convenient chemical groups and is meant to encompass any organic ions analysis of the proteins and / or lipid may be performed , e . g ., 10 that have two or more fluorine atoms, such as 3 or more , 4 mass spectroscopic analysis . or more , 5 or more, 6 or more , 7 or more , 8 or more , 9 or Ion Exchanger Composition more , or even 10 or more fluorine atoms. In some cases , the In the subject methods, the aqueous sample is subse - " fluorinated counterion ” includes a perfluoro group where quently contacted with an ion exchanger composition to all the hydrogens attached to carbon atoms of the group are produce a composition where the ionic liquid exchanges 15 replaced with fluorine atoms. As such , the " fluorinated cations with the ion exchanger composition in a salt metath - counterion ” may include one or more hydrocarbons ( i . e . , esis reaction . The term salt “metathesis ” reaction is used in C - H containing groups) . its conventional sense to refer to the transposition chemical In some embodiments , the ion exchanger counterion is process involving the exchange of bonds between two ionic described by the formula ( VII) : species which result in the exchanging of counterions 20 between the two salts . In other words , the subject ionic z '- L '- A -( - L2 - 22 ) liquid will undergo a metathesis reaction with the added salt wherein : to exchange counterions forming two new distinct salt Z is a perfluoroalkyl, an alkyl, a substituted alkyl, a compounds. This reaction may be represented generally by perfluoroaryl, an aryl or a substituted aryl; generic scheme 1 below : 25 n is 0 , 1, 2 or 3 ; each Z > , if present, is independently selected from the A - B + C - D - A - D + C - B ( Scheme 1) group consisting of a perfluoroalkyl, a perfluoroaryl, an In some embodiments , the ion exchange composition is a alkyl, a substituted alkyl , an aryl and a substituted aryl; second ionic liquid . For example , in certain embodiments, Ais a charged moiety capable of acting as a counterion to the ion exchange composition includes lithium bis [ ( trifluo - 30 the ionic liquid ; and romethane )sulfonyl ] imide (LiNTf2 ) and the anion exchange L ' and L are independently a covalent bond or a linker. with the ionic liquid includes the formation of a new ionic In certain embodiments , in formula (VII ) , Z ' is a perfluo salt having a bis [( trifluoromethane ) sulfonyl] imide (NT ) roalkyl, or a fluorinated alkyl. In certain embodiments , in anion . Any convenient ion exchanger compositions may be formula (VII ) , Z ' is a perfluoroaryl, or a fluorinated aryl. utilized in the subject methods . Ion exchanger compositions 35 In certain embodiments , in formula (VII ) , each Z2 is a of interest include those compositions described in U . S . perfluoroalkyl, or a fluorinated alkyl. In certain embodi provisional application Ser. No. 62 /051 , 804 , U . S . provi- ments , in formula (VII ) , each Z2 is a perfluoroaryl, or a sional application Ser . No . 62 /049 ,285 ; and U . S . provisional fluorinated aryl. application Ser. No. 14 / 205 , 100 , the disclosures ofwhich are In certain embodiments , in formula (VII ) , n is 0 . In certain incorporated herein by reference . 40 embodiments , in formula (VII ) , n is 1 . In certain embodi In some embodiments, the method includes combining an ments , in formula (VII ), n is 2 . In certain embodiments , in aqueous sample including an ionic liquid with an ion formula ( VII ) , n is 3 . exchanger composition including an ion exchanger counte - In certain embodiments , in formula (VII ), L ' and each L2 rion to produce a solution including a fluorous salt of the is an alkyl (e . g. , a linear alkyl linker , such as — CH2 - , ionic liquid . In the fluorous salt, at least one of the ionic 45 CH , CH , — or — (CH ), where m is an integer from 1 liquid and the ion exchanger counterion is fluorinated . As to 12 , e . g . , m is an integer from 1 to 6 ) . used herein , by “ fluorinated ” is meant that the group or In certain embodiments , in formula (VII ) , A is an organic moiety to which the term refers includes at least one fluorine anion selected from a sulfonimidate, a sulfonate , a carboxy substituent. In some cases , a fluorinated moiety is perfluo - late , a phosphate and a borate . rinated . As used here , the term “ perfluorinated ” refers to an 50 In certain embodiments , in formula (VII ) , when n = 1 , A is organic group or moiety that includes no C – H covalent SO , NOSO , — In certain embodiments, in formula bonds and a plurality of fluorinated substitutents . A perfluo (VII ) , when n = 0 , A is SO NHO ) . rinated group may include one or more additional non - In some embodiments , the perfluorous counterion is fluorine substituents . The ion exchanger composition may described by formula (VIII ) : include any convenient salt of the ion exchanger counterion . 55 In general terms, the ionic liquid combines with the ion z '- L '- A (VIII ) exchanger counterion to produce a fluorous salt that pro - wherein Z ' , L ' and A are as defined in for Formula (VII ) . vides for removal of the ionic liquid from the aqueous In certain embodiments , in formula (VIII ) , A is an organic sample . The ionic liquid and the ion exchanger counterion anion selected from a sulfonimidate , a sulfonate , a carboxy may be selected to provide for a salt having a desired 60 late , a phosphate and a borate . property . In certain embodiments , the salt is a fluorous salt In certain embodiments , in formulae (VII ) and (VIII ) , L ' that provides for extraction of the salt from the aqueous and each L ? are independently a C , - Co alkyl linker or a sample using a fluorous affinity substrate or a fluorous covalent bond . In certain embodiments , in formulae (VII ) solvent. In such cases, either the ionic liquid or the ion and (VIII ) , L ' and each L ? are independently selected from exchanger or both may be fluorinated in order to provide a 65 CH CH2 - or a covalent bond . fluorous salt that has affinity for a fluorous affinity substrate In certain embodiments , in formulae (VII ) and (VIII ) , Z and / or high solubility in a fluorous solvent . and Z ? are each independently a perfluoroalkyl group (e .g ., US 9 ,958 ,363 B2 20 comprising 4 or more fluorinated carbon atoms) . In certain embodiments , the ion exchanger composition includes a embodiments , in formulae ( VII ) and (VIII ) , Z and each Z2 silver salt of the ion exchanger counterion . In general terms, comprise together a combined total of 8 or more fluorinated the ion exchanger composition may include a salt of a metal carbon atoms ( e . g ., 10 or more , 12 or more , 14 or more , 16 cation and an ion exchanger counterion that is an anion . In or more , 18 or more or even 20 or more fluorinated carbon 5 the subject methods the ion exchanger counterion forms a atoms ) . new fluorous salt with the ionic liquid , and the metal cation In some embodiments, the ion exchanger counterion is an is free to form another salt with any convenient anions that ion exchanger counterion described by the formula ( IX ) : are present in the sample . The metal cation may be selected [Z ! — (CH2 ) m - S02 – N ( ) — SO2 – ( CH2) — Z ]. M * ( IX ) to provide for any desirable property , such as facile removal where : Z and Z are independently a perfluoroalkyl, an via an insoluble halide salt . In some embodiments , the alkyl, a substituted alkyl, a perfluoroaryl, an aryl, or a method further includes producing an insoluble silver salt , substituted aryl, wherein Z ' and Z ? include together a com such as silver chloride . In some instances , the method bined total of 8 or more fluorinated ( e . g . , perfluorinated ) further includes separating an insoluble silver salt from the carbon atoms (eg . 9 or more . 10 or more. 11 or more . 12 15 aqueous sample , where the silver cation of the salt is derived or more , 13 or more , 14 or more , 15 or more , 16 or more , from the ion exchanger composition . Any convenient meth 17 or more , 18 or more , 19 or more , 20 or more , 30 or more ods for separating a solid from a liquid may be utilized , or 40 or more fluorinated carbon atoms) ; m and p are including but not limited to , centrifugation , filtration , independently 0 , 1 or 2 ; and M + is a cation . decanting , and the like . In certain embodiments of formula (IX ) , Z ' and Z are the 20 Fluorous Salt same. In certain embodiments of formula (IX ) , Z ' and Z2 are Also provided is a composition including a fluorous salt the different . In certain embodiments of formula ( IX ) . Z ' and of an ionic liquid and a fluorous solvent . In some embodi z2 are each a fluorinated or perfluorinated group . In certain m ents of the composition , the fluorous salt of the ionic liquid embodiments of formula (IX ) , at least one of Z ' and Z2 is a includes a fluorinated ion exchanger counterion ( e . g . , as fluorinated or perfluorinated group . In certain embodiments 25 described herein ) and an ionic liquid cation ( e . g . , as of formula ( IX ) , Zl and Z2 are each a perfluoroalkyl. In described herein ). In some embodiments of the composition , certain embodiments of formula (IX ) . Z and Z2 are each the fluorous salt includes a fluorinated ion exchanger coun perfluorobutyl . In certain embodiments of formula ( IX ), z terion of formula (VII ) : [ Z ' - ( CH2) m _ SO2 - NO - S0 , and Z? are each perfluoropentyl. In certain embodiments of (CH2 ) . — Z? ] . M * ( VII) where : Z ' , Z?, m and p are as defined formula ( IX ) , Z and Z2 are each perfluorohexyl. In certain 30 above ; and M * is a cation selected from the group consisting embodiments of formula (IX ), Z ' and Z are each a perfluo of: roheptyl. In certain embodiments of formula ( IX ) , Z ' and 22 a ) Formula ( I) : are each a perfluorooctyl. In certain embodiments of formula (IX ) , Z ' and Z are each a perfluoroaryl. In certain embodi ments of formula ( IX ) , Z ' and Z2 include together a com - 35 bined total of 10 or more perfluorinated carbon atoms. In RI N- N - R2. certain embodiments of formula ( IX ) , Z and Z include together a combined total of 12 or more perfluorinated carbon atoms. In certain embodiments of formula (IX ), m wherein each of R and R is independently hydrogen , and p are each 0 . In certain embodiments of formula ( IX ), 40 alkyl, substituted alkyl, aryl, substituted aryl , arylalkyl , m + p = 1 . In certain embodiments of formula ( IX ) , m + p = 2 . In substituted arylalkyl, heteroalkyl , substituted heteroalkyl , certain embodiments of formula ( VII ), m + p = 3 . In certain heteroaryl, substituted heteroaryl, heteroarylalkyl, substi embodiments of formula ( IX ), m + p = 4 . In certain embodi- tuted heteroarylalkyl; ments of formula ( IX ) , M + is lithium . In certain embodi b ) Formula ( II ): ments of formula (IX ) , M + is potassium . In certain embodi- 45 ments of formula ( IX ) , M + is sodium . In certain embodiments of formula ( IX ) , M + is rubidium . In certain embodiments of formula (IX ), M + is silver. In certain instances , the ion exchange composition includes a salt having an anion selected from the group 50 Z consisting of boron tetrafluoride , bis - ( 2 , 4 , 4 -trimethylpentyl ) phosphinate , bis - ( trifluoromethyl) imide , bis [ ( trifluorometh ane ) sulfonyl] imide , bis - ( trifluoromethylsulfonyl) methane , bis -biphenyldiolatoborate , bis -malonatoborate , bis -oxalato wherein R is hydrogen , alkyl, substituted alkyl, aryl, borate , bis - (pentafluoroethyl ) phosphinate , bis - salicylatobo - 55 substituted aryl , arylalkyl, substituted arylalkyl, heteroalkyl, rate, bromine , butylsulfate , chloride , perchlorate , decanoate , substituted heteroalkyl, heteroaryl, substituted heteroaryl, dicyanamide, ethylsulfate , iodide, methylsulfate , octylsul heteroarylalkylsu , substituted heteroarylalkyl; fate , hexafluorophosphate , tetracyanoborate , toluene- 4 - sul c ) Formula ( III) : fonate , trifluoromethane -sulfonate , tris - ( nonafluorobutyl) trifluorophosphate and tris - ( pentafluoroethyl) 60 trifluorophosphate . In some embodiments , the ion exchanger counterion is bisnonafluoro - 1 - butanesulfonimidate . In some embodiments , the ion exchanger counterion is bis (( perfluo rohexyl) sulfonyl ) imide . R In some embodiments, the ion exchanger composition 65 includes a salt of the ion exchanger counterion selected from wherein R is hydrogen , alkyl, substituted alkyl, aryl, silver, lithium , sodium , rubidium and potassium . In some substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, US 9 , 958 , 363 B2 21 22 substituted heteroalkyl, heteroaryl, substituted heteroaryl, Fluorous Affinity Material heteroarylalkyl, substituted heteroarylalkyl; The subjectmethods include contacting the aqueous solu tion that includes the fluorous salt of the ionic liquid with a d ) Formula (IV ): fluorous affinity material. As used herein the term “ fluorous 5 affinity material” refers to a material, such as a support or a liquid that has greater affinity for a fluorinated moiety of interest than for a non - fluorinated moiety . In some instances, 2 the " fluorous affinity material” takes advantage of the heightened affinity that fluorinated moieties , such as per 10 fluorinated moieties , have for each other. The fluorous affinity material is capable of removing the fluorous salt from the aqueous solution . Any convenient fluorous affinity materials may be utilized in the subject methods to remove wherein R is hydrogen , alkyl, substituted alkyl, aryl , the fluorous salt from the aqueous solution . Fluorous affinity substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, 15 materials of interest include, but are not limited to , fluorous substituted heteroalkyl, heteroaryl, substituted heteroaryl, affinity chromatography supports such as FLUORO - PAKTM heteroarylalkyl, substituted heteroarylalkyl; and FLUORO - PAKTM II columns (Berry & Associates ) or e ) Formula ( V ) : fluorous silica , and fluorous solvents such as perfluorocar bons (PFCs ) and hydrofluoroethers (HFES ). Perfluorocar 20 bons of interest include , but are not limited to , perfluoro hexane , perfluoromethylcyclohexane and perfluorodecalin . R - NSle 1 Hydrofluoroethers of interest include , but are not limited to , nonafluorobutyl methyl ether ( e .g ., HFE -7100 ). In some embodiments , the fluorous solvent is methoxyperfluorobu 25 tane . wherein each of R1 and R2 is independently hydrogen , - Any convenient fluorous affinity methods and configura alkyl, substituted alkyl, aryl , substituted aryl, arylalkyl, tions of fluorous affinity materials may be utilized in the substituted arylalkyl, heteroalkyl, substituted heteroalkyl, removal of the fluorous salt from the aqueous sample . heteroaryl, substituted heteroaryl, heteroarylalkyl , substi Methods of interest include , but are not limited to , solid tuted heteroarylalkyl; and 30 phase extraction and liquid phase extraction , such as those f ) Formula ( VI) : methods described by Pearson et al. , “ Fluorous Affinity Purification of Oligonucleotides” , J . Org . Chem . , 2005 , 70 (18 ), pp 7114 -7122 ; US20060178507 ; Hayama et al. , Jour 7 nal of Pharmaceutical and Biomedical Analysis , Volume ©?? 35 101, December 2014 , Pages 151 - 160 ; Heitzman et al. , 7 " Fluorous ionic liquids as solvents for the liquid - liquid extraction of metal ions by macrocyclic polyethers ” , Tal wherein each of R1 and R2 is independently hydrogen , anta , Volume 69, Issue 2 , 15 Apr. 2006 , Pages 527 -531 ; and alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, Dandapani, S . , “ Recent applications of fluorous separation substituted arylalkyl, heteroalkyl, substituted heteroalkyl140, 4 methods in organic and bioorganic chemistry ” , QSAR & heteroaryl, substituted heteroaryl, heteroarylalkyl, substi Combinatorial Science 2006 , 25 , ( 8 - 9 ) , 681- 688 . After the fluorous salt has been removed from the aqueous tuted heteroarylalkyl. In some embodiments of the compo sample , e . g ., via solid - phase or liquid phase extraction , the sition , the cation is 1 -hexyl - 3 -methyl - imidazolium . In some remaining material of the aqueous sample may be referred to embodiments of the composition , the fluorinated ion 45 as an aqueous eluate . In some embodiments of the method , exchanger counterion is bis ( (perfluorohexyl ) sulfonyl) imide. “ the fluorous affinity material is an immiscible fluorous In some embodiments , the ionic liquid of the fluorous salt solvent that extracts the fluorous salt from the aqueous is fluorinated (e . g ., as described herein ). Any convenient solution to produce the aqueous eluate . In such cases , the fluorinated ionic liquid cations ( e . g ., as described herein ) aqueous eluate is not miscible with the fluorous solvent and may be utilized in the subject methods to produce a fluorous 50 may be easily separated from the fluorous solvent, e . g ., by salt. In certain cases , both the ionic liquid cation and the ion decanting. In some embodiments of the method , the fluorous exchanger counter ion of the fluorous salt are fluorinated affinity material is a fluorous affinity chromatography sup ( e .g ., as described herein ). In some embodiments , the ionic port that adsorbs the fluorous salt from the solution to liquid is not fluorinated . produce the aqueous eluate . In such cases , the aqueous In some instances, the ionic liquid exchanges anions with 55 eluate may be the solution that passes through the column the ion exchange composition to form a new salt having an and is collected . anion that is selected from boron tetrafluoride , bis- ( 2 ,4 , 4 - The aqueous eluate may be further analyzed using any trimethylpentyl) phosphinate , bis - ( trifluoromethyl) imide , bis convenient methods . Methods of analysis of interest include , [ ( trifluoromethane ) sulfonyl] imide , bis - ( trifluoromethylsul- but are not limited to , mass spectrometry (MS ) , gas chro fonyl) methane , bis - biphenyldiolatoborate , bis - 60 matography (GC ) , GC -MS , High performance liquid chro malonatoborate , bis - oxalatoborate , bis - ( pentafluoroethyl) matography (HPLC ) , HPLC -MS , capillary electrophoresis phosphinate , bis -salicylatoborate , bromine , butylsulfate , (CE ) , nuclear magnetic resonance (NMR ) , infrared spec chloride, perchlorate , decanoate , dicyanamide , ethylsulfate , troscopy , UV - vis spectroscopy, colorimetry, and the like . iodide , methylsulfate , octylsulfate , hexafluorophosphate , The aqueous eluate may include any convenient analytes of tetracyanoborate , toluene -4 -sulfonate , trifluoromethane -sul - 65 interest and the subject methods may provide for an fonate , tris - (nonafluorobutyl ) - trifluorophosphate and tris - improved detection and / or analysis of those analytes. In ( pentafluoroethyl) trifluorophosphate . some cases, the ionic liquid is a contaminant that prevents US 9 ,958 ,363 B2 23 24 sensitive detection and analysis of the analytes in the form ion cyclotron resonance (FTICR ), ion trap , quadrupole sample . For example , the ionic liquid may contaminate a or double focusing magnetic electric sector mass analyzers , mass spectrometer during sample analysis , e . g ., by suppress or any hybrid thereof. In one embodiment, the mass analyzer ing ion counts of an analyte of interest . In some embodi may be a sector, transmission quadrupole , or time -of - flight ments , the method further includes analyzing the aqueous 5 mass analyzer. eluate by mass spectrometry . In some cases , the removal of Methods for Preparation of a Cellular Sample for Analysis the ionic liquid from the aqueous sample leads to increased Also provided is a method for preparation of a cellular sensitivity of detection and /or analysis of an analyte by mass sample for analysis . In some cases , the method for prepa spectrometry . ration of a cellular sample for analysis includes extracting As described above, methods of the present disclosure 10 and purifying compounds from a biological sample includ may include analyzing the analyte containing compositions ing cells . The phrase " extracting and purifying” is used using liquid chromatography -mass spectrometry systems. herein in its conventional sense to refer to isolating desired For example , the apparatus may include analytical separa - compounds ( e . g . , metabolites ) from a plurality of compo tion device such as a liquid chromatograph (LC ) , including nents in a biological sample having cells . In some embodi a high performance liquid chromatograph (HPLC ) , a micro - 15 ments , the method further includes lysing cells of a biologi or nano - liquid chromatograph or an ultra -high pressure cal sample by contacting a biological sample with an amount liquid chromatograph (UHPLC ) device, a capillary electro - of the ionic liquid sufficient to denature intracellular meta phoresis (CE ) , or a capillary electrophoresis chromatograph bolic enzymes in the biological sample to produce the (CEC ) apparatus. However, any manual or automated injec aqueous sample. tion or dispensing pump system may be used . For instance , 20 In certain embodiments , compounds extracted by the a the subject sample may be applied to the LC -MS system subject methods are metabolites . The term “ metabolites ” is by employing a nano - or micropump in certain embodi - used herein its conventional sense to refer to one or more ments . compounds found which are the substrates or products of Mass spectrometer systems for use in the subject methods metabolic process which occur within a cell . As such , may be any convenient mass spectrometry system , which in 25 metabolites may include substrates or products which are general contains an ion source for ionizing a sample , a mass produced by metabolic processes including , but not limited analyzer for separating ions, and a detector that detects the to glycolysis, tricarboxylic acid cycle ( i. e ., TCA cycle , ions . In certain cases, the mass spectrometer may be a Krebs cycle ) , reductive pentose phosphate cycle ( i . e . , Calvin so - called “ tandem ” mass spectrometer that is capable of cycle ) , glycogen metabolism , pentose phosphate pathway , isolating precursor ions , fragmenting the precursor ions , and 30 among other metabolic processes . Accordingly , metabolites analyzing the fragmented precursor ions . Such systems are of interest may include but are not limited to glucose , well known in the art ( see, e . g ., U . S . Pat . Nos. 7 , 534 ,996 , glucose - 6 - phosphate , fructose -6 - phosphate , fructose - 1 , 6 7 ,531 ,793 , 7, 507 , 953 , 7 ,145 , 133 , 7 ,229 , 834 and 6 , 924 ,478 ) phosphate , glyceraldehyde 3 -phosphate , dihydroxyacetone and may be implemented in a variety of configurations. In phosphate , 1 , 3 -bisphosphoglycerate , 3 -phosphoglycerate , certain embodiments , tandem mass spectrometry may be 35 2 -phosphoglycerate , phosphoenolpyruvate , pyruvate , acetyl done using individual mass analyzers that are separated in COA , citrate , cis -aconitate , d - isocitrate , a -ketoglutarate , space or, in certain cases , using a single mass spectrometer succinyl CoA , succinate , fumarate , malate , oxaloacetate , in which the different selection steps are separated in time. ribulose 1 , 5 -bisphosphate , 3 -phosphoglycerate , 1 , 3 - bispho Tandem MS “ in space” involves the physical separation of sphoglycerate , glyceraldehyde 3 -phosphate , ribulose -5 the instrument components ( QqQ or ( TOF) whereas a 40 phosphate , ethanol, acetylaldehyde, pyruvic acid , 6 -phos tandem MS " in time” involves the use of an ion trap . phogluconolactone, 6 - phosphogluconate , ribose - 5 An example mass spectrometer system may contain an phosphate , xylulose - 5 -phosphate , sedoheptulose ion source containing an ionization device , a mass analyzer 7 - phosphate , erythrose 4 -phosphate , among other metabo and a detector. As is conventional in the art, the ion source lites . and the mass analyzer are separated by one or more inter - 45 Aspects of the method include lysing cells of a biological mediate vacuum chambers into which ions are transferred sample and contacting the biological sample with an amount from the ion source via , e . g . , a transfer capillary or the like . of ionic liquid sufficient to denature intracellular metabolic Also as is conventional in the art, the intermediate vacuum enzymes in the biological sample . By “ lyse " cells is meant chamber may also contain a skimmer to enrich analyte ions that the cells are ruptured or broken open such that the ( relative to solvent ions and gas ) contained in the ion beam 50 internal contents of the cells , including metabolic enzymes exiting the transfer capillary prior to its entry into the ion are released into the surrounding medium ( e . g ., ionic liquid ) . transfer optics ( e . g ., an ion guide, or the like) leading to a In some embodiments , cell lysis may further include lysis of mass analyzer in high vacuum . cellular organelles , for example the nucleus, mitochondria , The ion source may rely on any type of ionization method , ribosomes , chloroplasts, lysosomes, vacuoles, Golgi appa including but not limited to electrospray ionization (ESI ) , 55 ratus, centrioles, etc . such that the contents of the cellular atmospheric pressure chemical ionization ( APCI) , electron organelles are also released into the surrounding medium . impact (EI ) , atmospheric pressure photoionization (APPI ), In some embodiments , lysing the cells of a biological matrix - assisted laser desorption ionization (MALDI ) or sample is performed by contacting the cells of the biological inductively coupled plasma ( ICP ) ionization , for example , or sample with a lysing agent ( e . g . , an ionic liquid , as described any combination thereof ( to provide a so - called " multi- 60 herein ) . The lysing agent may be any suitable lysing agent mode ” ionization source ) . In one embodiment, the precursor so long as it is sufficient to break open the cells where that ions may be made by EI, ESI or MALDI, and a selected the internal contents of the cell are released into the sur precursor ion may be fragmented by collision or using rounding medium . The lysing agent may be contacted with photons to produce product ions that are subsequently the biological sample having the cells at the same time (i . e . , analyzed . 65 simultaneously ) as contacting the biological sample having Likewise , any of a variety of differentmass analyzers may cells with ionic liquid . In some cases , the lysing agent may be employed , including time of flight ( TOF ) , Fourier trans - be contacted with the biological sample sufficient to break US 9 ,958 ,363 B2 25 26 open the cells before contacting the sample with the ionic glyceraldehyde 3 - phosphate dehydrogenase, phosphopen liquid . In other words, in these embodiments , the biological tose epimerase, phosphoribulokinase , glucose -6 -phosphate sample having cells that is contacted with the ionic liquid dehydrogenase , gluconolactonase , 6 - phosphogluconate includes cells which have been previously broken open by dehydrogenase , ribulose - 5 -phosphate isomerase, ribulose - 5 one or more lysing agents . In certain embodiments , the ionic 5 phosphate 3 - epimerase , transaldolase , transketolase , among liquid functions as the lysing agent and contacting the other metabolic pathway enzymes. biological sample having cells with the ionic liquid is Contacting the biological sample having cells with the sufficient to lyse the cells of the sample and denature ionic liquid may include mixing the cells in the ionic liquid . intracellular metabolic enzymes without the need for an Any convenient method may be employed to stir the bio additional lysing agent. In some embodiments , the method 10 logical sample having cells with the ionic liquid , so long as includes contacting a biological sample having cells with an the cells are sufficiently mixed throughout and in contact amount of ionic liquid composition ( e . g . , as described with the ionic liquid . Mixing may include , for example herein ) sufficient to lyse the cells and denature intracellular stirring with a magnetic stir bar or manually stirred using metabolic enzymes in the biological sample . In certain any convenient stirring apparatus. Alternatively, the biologi instances of the method , the ionic liquid composition is an 15 cal sample in the ionic liquid may be stirred by vortexing the aqueous composition including 30 % or more of an ionic contacted sample , shaking the contacted sample such as with liquid by weight, such as 35 % or more by weight, 40 % ora mechanical shaker or shaking may be manually performed more by weight, 45 % or more by weight, 50 % or more by ( i. e ., by hand ). In some instances , mixing the biological weight, 55 % or more by weight, 60 % or more by weight , sample having cells with the ionic liquid includes sonicating 65 % or more by weight, 70 % or more by weight, 75 % or 20 the contacted composition . more by weight , 80 % or more of an ionic liquid by weight, As described above , methods include contacting a bio or even more . logical sample having cells with an ionic liquid . As noted As described in greater detail below , intracellular above, ionic liquids of interest destabilize , disrupt or dena enzymes are denatured by contacting with the ionic liquid . ture enzyme structure . Any convenient ionic liquid may be The term " denature ” is used in its conventional sense to 25 employed in the subject methods so long as the ionic liquid mean that the structural conformation of the subject proteins destabilizes , disrupts and / or denatures enzyme structure . or enzymes is destabilized or disrupted , in certain embodi- Contacting the biological sample having cells with an ments the proteins or enzymes losing quaternary , tertiary and ionic liquid lyses the cells , releasing the cellular components secondary structure that is otherwise present in its native into the ionic liquid and denatures the metabolic enzymes state . Protein denaturation by the ionic liquid includes 30 found within the cells. As such , contacting the biological quaternary denaturation where protein sub - units are disso sample having cells according to the subject methods ciated or the spatial arrangement of protein subunits is quenches metabolic processes of the cell upon contact/ disrupted . Protein denaturation by ionic liquids may further mixing with the ionic liquid . By “ quenches metabolic pro include tertiary structure denaturation which includes the cesses” is meant that metabolic processes which occur in the disruption of covalent interactions between amino acid side 35 native cell are stopped by lysis and denaturation . As such , in chains (such as disulfide bridges between cysteine groups ) , practicing the subject methods , 95 % or more of metabolic non - covalent dipole -dipole interactions between polar process in the cell may be quenched after contacting the amino acid side chains and surrounding media , Van der biological sample with the ionic liquid , such as 97 % or Waals interactions ( e . g ., induced dipole moments ) between more , 99 % or more , 99 . 5 % or more , or 99 . 9 % or more , and non - polar amino acid side chains . Protein denaturation by 40 including all of the metabolic processes mediated by ionic liquids may further include secondary structure dena - enzymes which are quenched by lysis and denaturation of turation where the proteins or enzymes lose all regular metabolic enzymes by contacting with the ionic liquids . repeating patterns such as alpha -helices and beta -pleated In some instances of the method , reverse phase chroma sheets and may adopt a random - coil type configuration . In tography is used to adsorb proteins and /or lipids thatmay be some embodiments , the biological sample having cells is 45 present in the aqueous cellular sample . In some embodi contacted at room temperature (i . e . , about 20° C . or 68° F . ments , the method further includes contacting the aqueous or 293K ). cellular sample including an ionic liquid with a reverse Where compounds extracted and purified by the subject phase substrate , thereby adsorbing proteins and / or lipids on methods include metabolites , ionic liquids of interest the reverse phase substrate . As such , the cellular sample may include those sufficient to destabilize , disrupt or denature 50 be one to which an ionic liquid has been added to lyse the metabolic enzymes . Metabolic enzymes of interest include , cells and / or quench metabolism of the cells in the sample . In but are not limited to those employed in the metabolic such cases , removal of proteins and / or lipids from the processes discussed above , such as metabolic enzymes in sample ensures metabolism of the cell remains quenched in glycolysis , tricarboxylic acid cycle ( i. e . , TCA cycle , Krebs the aqueous sample , e . g . , by removing metabolic enzymes cycle ) , reductive pentose phosphate cycle ( i. e . , Calvin 55 from analytes of interest . cycle ) , glycogen metabolism , the pentose phosphate path In some embodiments , after contacting the biological way, among other metabolic processes. For example , meta sample with the ionic liquid , the cellular sample is mixed bolic enzymes of interest include , but are not limited to : with an organic solvent . In certain embodiments , the organic hexokinase, phosphoglucose isomerase , phosphofructoki- solvent may be added to the ionic liquid cellular sample to nase , fructose bisphosphate aldolase , triose phosphate 60 form an ionic liquid cellular sample - organic solvent two isomerase , glyceraldehyde phosphate dehydrogenase , phase composition . By “ two phase composition ” is meant phophoglycerate kinase , phosphoglycerate mutase, enolase , that the ionic liquid cellular sample is not miscible with the pyruvate kinase , pyruvate dehydrogenase , citrate synthase , organic solvent and forms two distinct layers . As such , in aconitase , isocitrate dehydrogenase , a -ketoglutarate dehy - these embodiments , the organic solvent and ionic liquids are drogenase , succinyl - CoA synthetase , succinic dehydroge - 65 not miscible . For example , where the ionic liquid is hydro nase , fumarase , malate dehydrogenase , pyruvate carboxy - philic , the organic solvent may be hydrophobic . Likewise , lase , ribulose - 1 ,5 - bisphophate carboxylase oxygenase , where the ionic liquid is hydrophobic , the organic solvent US 9 ,958 ,363 B2 27 28 may be hydrophilic . In certain embodiments where a two - composition where the ionic liquid exchanges cations with phase composition is formed , the organic liquid is denser the ion exchange composition in a salt metathesis reaction . than the ionic liquid . In other words, after addition of the In embodiments of the invention , the addition of the ion organic solvent to the ionic liquid , the organic phase is exchanger composition to the dispersed microdroplet com positioned at the bottom of the two -phase composition and 5 position is sufficient to form an ionic liquid cellular sample the ionic liquid cellular sample phase is positioned on top . organic solvent two phase composition . As noted above, the In some embodiments , the organic liquid mixed with the two phase composition includes an ionic liquid composition that is not miscible with the organic solvent and thus , forms ionic liquid cellular sample is a hydrophobic or non -polar two distinct layers . By contacting the dispersed microdroplet organic solvent. Hydrophobic or non - polar organic solvents 10 composition with the ion exchange composition , the organic of interest include, but are not limited to , fluorous solvents , layer and ionic liquid layers can be separated . In certain pentane , hexane , heptane , octane , diethyl ether, and chloro embodiments , the newly formed ionic liquid with exchanged form . Where a hydrophobic or non - polar organic solvent is anion is denser than the organic solvent layer . In some cases, employed , non - polar and hydrophobic cellular components after addition of the ion exchange composition to the dis may be extracted into the organic solvent layer. As such , the 15 persed microdroplet composition , the organic solvent phase hydrophobic cellular components ( e . g . , lipids, nonpolar is positioned at the top of the two -phase composition and the membrane components , etc . ) from the ionic liquid cellular ionic liquid phase is positioned on the bottom . sample are extracted into the organic phase of the two phase After formation of distinct layers in an ionic liquid composition . organic solvent two -phase composition , the ionic liquid may In certain embodiments , a dispersed microdroplet com - 20 be separated from the organic solvent . The ionic liquid may position is produced after mixing an organic liquid with the be separated from the organic layer by any convenient ionic liquid cellular sample . The term “ microdroplet ” is used protocol, including but not limited pouring off the organic in its conventional sense to refer to aggregates of the ionic solvent, aspirating to separate the ionic liquid from the liquid cellular sample composition within the organic sol- organic solvent ( e . g . , using either a manual, mechanically vent medium having dimensions ranging from 0 . 001 um to 25 controlled , hydraulically controlled or electrically controlled 1000 um , such as 0 .01 um to 100 um , such as 0 . 1 um to 10 pipet ) or by evaporation of the organic solvent ( e . g ., vacuum um and including 1 um . By forming the microdroplets , the evaporation , by bubbling inert gas through the organic surface area of the ionic liquid cellular sample is increased , phase ). where in certain instances the hydrophobic components in Methods of the present disclosure may further include the ionic liquid cellular sample are extracted into the organic 30 separating the target compounds ( e . g . , metabolites ) solvent medium . Likewise , by forming a dispersed micro - extracted from the biological sample cells from the ionic droplet composition , the proteins and enzymes denatured by liquid , such as for example by microextraction . Microex contacting with the ionic liquid precipitate . In some case , traction protocols of interest may be any convenient micro remaining in the ionic liquid phase are the subject com extraction so long as the protocol is sufficient to extract the pounds , e . g . , metabolites . In certain cases , the subject com - 35 target metabolites from the ionic liquids . For example , pounds, e . g . , metabolites, are dissolved in the aqueous microextraction may include solid phase chromatography. In phase . certain embodiments , solid phase chromatography includes , Microdroplet dispersions may be formed using any con - but is not limited to ion exchange chromatography, liquid venient protocol, so long as the ionic liquid cellular sample chromatography employing a reverse phase stationary col organic solvent composition is agitated sufficiently to form 40 umn , among other chromatography protocols. dispersed microdroplets of ionic liquid cellular sample in the In some embodiments , separating the metabolites from organic solvent medium . In certain embodiments , agitation the ionic liquid further includes analysis of the separated may result in turbid solutions having a plurality of micro - metabolites . By analyzed is meant characterizing the chemi droplets homogeneously dispersed throughout the organic cal composition of the separated metabolites, including but solvent . Agitation may include , but is not limited to , vor- 45 not limited to the amount and types of compounds in the texing the composition , sonicating the composition , shaking extracted metabolites as well as any impurities present. the composition either manually ( i. e . , by hand ) or mechani- Chemical analysis may be conducted using any convenient cally ( i . e . , by a mechanically or electrically powered shaking protocol, such as for example by mass spectrometry , infrared device ) , and rapidly stirring the composition manually , spectroscopy, UV -vis spectroscopy , colorimetry and nuclear among other agitating protocols . Agitation may be per - 50 magnetic resonance spectroscopy . In certain embodiments , formed for any amount of time, so long as agitation is chemical analysis is conducted by gas chromatography sufficient to produce the desired microdroplet dispersions. mass spectrometry . In other embodiments , chemical analysis As such , agitation may be performed for one second or is conducted by liquid chromatography -mass spectrometry . longer , such as for two seconds or longer, such as for 5 Ionic liquids are suitable for denaturing proteins and have seconds or longer , such as for 10 seconds or longer , for 30 55 been used for the extraction of small molecules and DNA . seconds or longer, for 1 minute or longer , for 5 minutes or Using an ionic liquid , the entire cellular contents of a longer , or for 10 minutes or longer and including agitation biological sample can be solubilized and denatured . By for 30 minutes or longer. denaturing the proteins, the degradation of DNA and RNA In certain embodiments , a dispersant is not added to the can be significantly reduced . In one embodiment, an ionic composition to produce the microdroplet dispersions. As 60 liquid can be used to solubilize the entire contents of a such , in these embodiments no additional compounds are biological sample, denature proteins, DNA , and RNA , and added to the composition in order to create the microdroplet separate each individual component from the mixture . dispersions other than agitation of the sample . The denaturation process can be instantaneous when a In some instances of the subject methods , the dispersed large amount of ionic liquid is introduced to the sample ( e . g . , microdroplet composition may be subsequently contacted 65 at least 2x , at least 5x or at least 10x , by volume) . After with an ion exchanger composition ( e . g ., as described addition of the ionic liquid , proteins and / or lipids can be herein ) to produce an ionic liquid -organic solvent two phase removed from the sample , e .g ., via an amine reactive moiety US 9 ,958 ,363 B2 29 30 that is attached to a solid phase or by reverse phase chro - ( e . g ., a cation of one of formulae ( I) to (VI ) , as described matography. By removing the protein and / or lipids from the herein ) and a fluorinated ion exchanger counterion ( e . g . , a sample , the remaining components may be much less sus counterion of formula (VII ) , as described herein ) . In some ceptible to degradation . In certain cases , a cleavable linker cases, the fluorinated ion exchanger counterion is bis ( ( per may be attached to the solid phase so that the proteins can 5 fluorohexyl sulfonyl ) imide . In some instances , the ionic be released from the solid phase and later analyzed by mass liquid cation is 1 - hexyl- 3 -methyl - imidazolium . In certain spectrometry . embodiments , the fluorous solvent is methoxyperfluorobu In some embodiments , the method includes combining an tane . The fluorous salt of the ionic liquid may have a ionic liquid with a sample and removing the protein and /or partition coefficient in the fluorous solvent ( e. g . , HFE -7100 ) , lipid from the sample using a reverse phase chromatography 10 relative to water of 99 . 9 % or more , such as 99 . 95 % or more , support to produce an aqueous sample . In certain cases , the 99. 98 % or more , 99 .99 % or more, 99 . 995 % or more , protein may be released from the support and analyzed . 99 . 998 % or more , or 99 . 999 % or more . In some embodi Following the optional removal of the proteins and / or lipids , ments , the ion exchanger composition is soluble ( e . g ., spar an ion -exchange reaction can be induced by adding an ion ingly soluble ) in water. In some embodiments , the ion exchanger composition ( e . g ., as described herein ) . 15 exchanger composition has a partition coefficient in the In some instances , the method includes combining the fluorous solvent ( e . g . , HFE - 7100 ) , relative to water of aqueous sample either sequentially or simultaneously with : 99 . 8 % or less , such as 99 % or less , 95 % or less , 90 % or less , ( i ) an ion exchanger composition comprising a fluorinated 85 % or less or 80 % or less , but as the metathesis reaction ion exchanger counterion to produce a fluorous salt of the occurs , the fluorinated ion exchanger counterion forms a ionic liquid ; and ( ii ) an immiscible fluorous solvent . The ion 20 new fluorous salt with the ionic liquid cation that has an exchanger composition and the fluorous solvent may be increased partition coefficient in the fluorous solvent ( e . g . , as added to the aqueous sample in any convenient order and described herein ) . using any convenient method . The fluorous solvent is not Kits and Systems miscible with the aqueous sample and forms a separate Also provided by this disclosure are kits for practicing the liquid phase into which the fluorous salt of the ionic liquid 25 subject method as described above . In some instances, the dissolves. The partition coefficient of the fluorous salt in the kit includes an ion exchanger composition ( e . g . , as described aqueous sample versus the fluorous solventmay be selected herein ) and a fluorous solvent. In certain instances of the kit , so as to provide for a desired level of extraction of the ionic the ion exchanger composition includes a fluorinated ion liquid from the aqueous sample . exchanger counterion ( e .g ., a counterion of formula (VII ) , as After formation of distinct layers in an aqueous sample - 30 described herein ) . fluorous solvent two -phase composition , the ionic liquid A subject kit may contain one or more of: an ionic liquid may be separated by any convenient protocol , including but in an amount sufficient to lyse cells and denature intracel not limited pouring off the fluorous solvent , aspirating to lular metabolic enzymes in the biological sample ; a protein separate the aqueous eluate from the fluorous solvent ( e . g ., and / or lipid adsorbing matrix selected from a reverse phase using either a manual, mechanically controlled , hydrauli - 35 matrix , an ion exchange matrix and a size exclusion matrix ; cally controlled or electrically controlled pipet) . In some and instructions for extracting metabolites from the cells of instances, the method further includes collecting the aque - the biological sample . In some embodiments of the kit , the ous phase of the two phase system to produce an aqueous ionic liquid is comprised in an aqueous composition includ eluate . ing 30 % or more of the ionic liquid by weight . As such , the fluorous salt is extracted from the aqueous 40 In some embodiments , the kit is a kit for extracting sample into the fluorous affinity liquid to produce an aque - metabolites from a biological sample . The kit may include : ous eluate of the remaining material of the aqueous cellular a n ionic liquid in an amount sufficient to lyse cells and sample . The steps of this extraction process may be repeated denature intracellular metabolic enzymes in the biological one ormore times, such as two or more, 3 or more, 4 or more sample ( e . g . , as described herein ) ; and an ionic exchange or 5 or more times , as desired to remove any remaining ionic 45 composition including a fluorinated ionic exchange counte liquid from the aqueous sample . Any convenient number of rion ( e . g . , as described herein ) . In some embodiments of the extractions may be performed as necessary to ensure that a kit , the ionic liquid is comprised in an aqueous composition desired level of fluorous salt is achieved in the resulting including 30 % ormore of the ionic liquid by weight. In some aqueous eluate . In certain embodiments , the aqueous eluate embodiments of the method , the ionic liquid includes that is produced after liquid phase extraction may be further 50 1 -hexyl - 3 -methyl - imidazolium . In certain instances, the kit contacted with a solid phase fluorous affinity support, further includes one or more components selected from the thereby absorbing residual fluorous salt of the ionic liquid group consisting of: a fluorous solvent; a protein and /or lipid from the aqueous eluate , and producing an aqueous eluate adsorbing matrix selected from a reverse phase matrix , an substantially free from ionic liquid . By substantially free ion exchange matrix and a size exclusion matrix ; and from ionic liquid is meant a solution that includes 0 . 5 % or 55 instructions for extracting metabolites from the cells of the less by weight of the ionic liquid , such as 0 . 1 % or less by biological sample . weight, 0 .03 % or less by weight, 0 .01 % or less by weight, The kitmay also include containers , measurement devices 0 .003 % or less by weight, 0 .001 % or less by weight , and instruments for performing the subject methods, e . g . , 0 .0003 % or less by weight, or 0 .0001 % or less by weight. vials , agitators , shakers , vortexers , pipets , filter membranes , Compositions 60 etc . The various components of the kit may be present in Aspects of the invention further include a composition separate containers or certain compatible components may including : a fluorous salt of an ionic liquid (e . g ., as described be pre - combined into a single container, as desired . In herein ) ; and a fluorous solvent ( e. g . , as described herein ) . certain instances of the kit , the ionic exchange composition The fluorous salt of the ionic liquid may be dissolved in the is disposed in a plurality of containers . In certain instances fluorous solvent. The fluorous salt of ionic liquid may 65 of the kit , the protein and /or lipid adsorbing matrix is include a fluorinated ion exchanger counterion . In some disposed in a plurality of containers. Any convenient con instances, the fluorous salt is a salt of an ionic liquid cation tainers may be utilized , including single containers such as US 9 ,958 ,363 B2 31 32 vials , tubes, or bottles , and multi- well containers, such as condition ( where the presence ofmetabolic profile is indica multi -well plates, multiplexed tubes, filter tubes, etc . In tive of a disease or condition ) , discovery of drug targets some cases, the plurality of containers is a multiwell plate . (where , e . g ., ofmetabolic profile associated with a disease or In addition to above -mentioned components , the subject condition and may be targeted for drug therapy ) , drug kits may further include instructions for using the compo - 5 screening (where the effects of a drug are monitored by nents of the kit to practice the subject methods , i . e ., to assessing a metabolic profile ) , determining drug suscepti provide instructions for sample analysis . The instructions for bility (where drug susceptibility is associated with a par practicing the subject methods are generally recorded on a ticular metabolic profile ) and basic research ( where is it suitable recording medium . For example , the instructions desirable to identify the a metabolic profile in a sample , or, may be printed on a substrate , such as paper or plastic , etc . 10 in certain embodiments , the relative levels of a particular As such , the instructions may be present in the kits as a metabolites in two or more samples ) . package insert, in the labeling of the container of the kit or In certain embodiments , relative levels of a set of metabo components thereof ( i . e . , associated with the packaging or lites in two or more different nucleic acid samples may be subpackaging ) etc . In other embodiments , the instructions obtained using the above methods , and compared . In these are present as an electronic storage data file present on a 15 embodiments , the results obtained from the above - described suitable computer readable storage medium , e . g . , CD -ROM , methods are usually normalized to the total amount of a diskette , etc . In yet other embodiments , the actual instruc control metabolite , and compared . This may be done by tions are not present in the kit , but means for obtaining the comparing ratios , or by any other means . In particular instructions from a remote source , e .g ., via the internet, are embodiments , the nucleic acid profiles of two or more provided . An example of this embodiment is a kit that 20 different samples may be compared to identify metabolites includes a web address where the instructions can be viewed that are associated with a particular disease or condition . and /or from which the instructions can be downloaded . As In some examples , the different samples may consist of an with the instructions, this means for obtaining the instruc “ experimental” sample , i. e ., a sample of interest, and a tions is recorded on a suitable substrate . " control” sample to which the experimental sample may be Also provided is a system for analytical sample treatment, 25 compared . In many embodiments , the different samples are including a container having disposed therein : an ion pairs of cell types, one cell type being a cell type of interest , exchanger composition ; and a fluorous solvent. In some e .g . , an abnormal cell, and the other a control, e . g ., normal, embodiments of the system , the ion exchanger composition cell. If two fractions of cells are compared , the fractions are includes a fluorinated ion exchanger counterion . In certain usually the same fraction from each of the two cells . In instances, the system includes a plurality of the containers , 30 certain embodiments , however, two fractions of the same each container having disposed therein the ion exchanger cell may be compared . Exemplary cell type pairs include, for composition and the fluorous solvent. In some cases , the example , cells that are treated ( e .g ., with environmental or plurality of containers is a multiwell plate . Any convenient chemical agents such as peptides, hormones , altered tem containers may be utilized in the subject systems, including perature , growth condition , physical stress, cellular trans single containers such as vials, tubes , or bottles, and mul - 35 formation , etc . ), and a normal cell ( e . g ., a cell that is tiwell containers , such as multiwell plates (e . g ., 96 -well otherwise identical to the experimental cell except that it is plates) , multiplexed tubes, filter tubes, etc . not immortal, infected , or treated , etc . ) ; cells isolated from Utility a tissue biopsy ( e . g ., from a tissue having a disease such as The method , composition , system and kit described above colon , breast , prostate , lung, skin cancer , or infected with a may be used to analyze analytes of interest in any of a 40 pathogen etc .) and normal cells from the same tissue, usually variety of different samples , including metabolites in cellu from the same patient; cells grown in tissue culture that are lar sample , proteins in proteomics samples, and lipids in immortal ( e . g . , cells with a proliferative mutation or an biological samples. immortalizing transgene ) , infected with a pathogen or a cell Cellular samples of interest include bacterial cells such as isolated from a mammalwith a cancer, a disease , a geriatric E . coli cells , and eukaryotic cells such as cells of a lower 45 mammal, or a mammal exposed to a condition , and a cell eukaryote , e .g ., yeast , or a higher eukaryote such as a plant from a mammal of the same species , preferably from the ( e . g ., monocot or dicot) or an animal ( e . g . , an insect, same family , that is healthy or young ; and differentiated cells amphibian , or mammalian etc. ) . In certain cases , the source and non -differentiated cells from the samemammal ( e . g . , of the cells may or may not have a cell wall , and in certain one cell being the progenitor of the other in a mammal , for embodiments , the cells may be photosynthetic or non - 50 example ) . photosynthetic , oleaginous or non - oleaginous. In particular embodiments , the cells are not algae . The cells may be EMBODIMENTS cultured cells , or, in certain embodiments , cells from a tissue. Aspects of the present disclosure include a method for The method described above may be used for metabolo - 55 removing an ionic liquid from an aqueous sample . In some mics studies , i . e . , systematic studies of the unique chemical embodiments , the method includes: ( a ) combining an aque fingerprints that are associated with specific cellular pro ous sample including an ionic liquid with an ion exchanger cesses and the study of their metabolite profiles . The composition including an ion exchanger counterion to pro metabolome represents the complete set of small -molecule duce a solution including a fluorous salt of the ionic liquid , metabolites ( such as metabolic intermediates, hormones and 60 wherein at least one of the ionic liquid and the ion exchanger other signaling molecules , and secondary metabolites ) to be counterion is fluorinated ; (b ) contacting the solution with a found within a biological sample , such as a single organism . fluorous affinity material, thereby removing fluorous salt The subject method , composition , system and kit may be from the solution and producing an aqueous eluate ; and (c ) employed in a variety of drug discovery , research and collecting the aqueous eluate . In some embodiments of the diagnostic applications. For example , a subject method may 65 method , the fluorous affinity material is an immiscible be employed in a variety of applications that include , but are fluorous solvent that extracts the fluorous salt from the not limited to , diagnosis or monitoring of a disease or solution to produce the aqueous eluate . In some embodi US 9 , 958 , 363 B2 33 34 ments of the method , the fluorous affinity material is a eroalkyl, substituted heteroalkyl, heteroaryl , substituted het fluorous affinity chromatography support that adsorbs the eroaryl, heteroarylalkyl, substituted heteroarylalkyl; and fluorous salt from the solution to produce the eluate . f ) Formula ( VI) : In some embodiments of the method , the ionic liquid includes a cation selected from the group consisting of: 5 a ) Formula (I ) : R2 R N- N - R2, 10 wherein each of R and R is independently hydrogen , alkyl, substituted alkyl , aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, wherein each of R1 and R2 is independently hydrogen , heteroaryl, substituted heteroaryl, heteroarylalkyl, substi alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, 1 tuted heteroarylalkyl . In some embodiments , the ionic liquid substituted arylalkyl, heteroalkyl, substituted heteroalkyl, includes 1 -hexyl - 3 -methyl - imidazolium . heteroaryl , substituted heteroaryl, heteroarylalkyl, substi In some embodiments of the method , the ion exchanger tuted heteroarylalkyl; counterion is a fluorinated counterion described by the b ) Formula ( II ) : formula (VII ) : [ Z ( CH2) m SO , NOSO (CH2 ) , — 20 Z2] . M * (VII ) where : 27 and are independently a per fluoroalkyl, an alkyl, a substituted alkyl, a perfluoroaryl, an aryl, or a substituted aryl, wherein Z and Z2 include together a combined total of 8 or more fluorinated carbon atoms; m and p are independently 0 , 1 or 2 ; and M + is a Z 25 cation . In some embodiments of the method , the ion exchanger counterion is bisnonafluoro - 1 - butanesulfonimi date . In some embodiments of the method , the ion exchanger counterion is bis ( ( perfluorohexyl sulfonyl) imide . In some wherein R is hydrogen , alkyl, substituted alkyl, aryl, embodiments of the method , the ion exchanger composition substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, 30 includes a salt of the ion exchanger counterion selected from substituted heteroalkyl, heteroaryl, substituted heteroaryl, silver, lithium , sodium and potassium . In some embodiments heteroarylalkyl, substituted heteroarylalkyl; of the method , the ion exchanger composition includes a c ) Formula ( III ) : silver salt of the ion exchanger counterion . In some embodi ments of the method , step ( a ) further includes producing an 35 insoluble silver salt . In some embodiments , the method further includes, prior to step (a ), contacting the aqueous sample including an ionic liquid with a reverse phase substrate , thereby adsorbing proteins and / or lipids on the reverse phase substrate , if 40 present in the aqueous sample . In some embodiments , the wherein R is hydrogen , alkyl, substituted alkyl, aryl, method further includes analyzing the eluate by mass spec substituted aryl, arylalkyl , substituted arylalkyl, heteroalkyl, trometry . substituted heteroalkyl, heteroaryl , substituted heteroaryl, In some embodiments , the method further includes : lysing heteroarylalkyl , substituted heteroarylalkyl; cells of a biological sample; and contacting a biological d ) Formula (IV ) : 45 sample with an amount of the ionic liquid sufficient to denature intracellular metabolic enzymes in the biological sample to produce the aqueous sample . Also provided is a method for preparation of a cellular sample for analysis . In some embodiments , the method A2+ 50 includes : ( a ) contacting a cell with an amount of an ionic liquid composition sufficient to lyse the cell and produce an aqueous sample including an ionic liquid ; ( b ) contacting the aqueous sample with a reverse phase substrate , thereby adsorbing proteins and/ or lipids of the cell on the reverse wherein R is hydrogen , alkyl, substituted alkyl, aryl, 55 phase substrate and producing a contacted aqueous sample ; substituted aryl, arylalkyl , substituted arylalkyl, heteroalkyl, ( c ) combining the contacted aqueous sample either sequen substituted heteroalkyl, heteroaryl, substituted heteroaryl, tially or simultaneously with : ( i ) an ion exchanger compo heteroarylalkyl, substituted heteroarylalkyl; sition including a fluorinated ion exchanger counterion to e ) Formula ( V ) : produce a fluorous salt of the ionic liquid ; and (ii ) an immiscible fluorous solvent, thereby extracting the fluorous salt into the fluorous affinity liquid and producing an aque ous eluate ; and ( d ) collecting the aqueous eluate of step (C ); R - N and optionally repeating step (c ) one or more times on the aqueous eluate until a desired level of fluorous salt in the 65 aqueous eluate is achieved . wherein each of R is hydrogen , alkyl , substituted alkyl, In some embodiments , the method further includes con aryl, substituted aryl, arylalkyl, substituted arylalkyl, het - tacting the aqueous eluate with a fluorous affinity chroma US 9 ,958 ,363 B2 35 36 tography support, thereby adsorbing residual fluorous salt wherein R is hydrogen , alkyl, substituted alkyl, aryl, from the aqueous eluate. In some embodiments , the method substituted aryl , arylalkyl, substituted arylalkyl, heteroalkyl, further includes analyzing the aqueous eluate by mass substituted heteroalkyl, heteroaryl, substituted heteroaryl, spectrometry . In some embodiments of the method , the ionic liquid composition is an aqueous composition including 5 heteroarylalkyl, substituted heteroarylalkyl; 30 % or more of an ionic liquid . In some embodiments of the d ) Formula (IV ) : method , the ionic liquid includes 1 -hexyl - 3 -methyl - imida zolium . In some embodiments of the method , the ion exchanger composition includes a silver salt of the fluori nated ion exchanger counterion . In some embodiments of the method , the fluorinated ion exchanger counterion is -zl+ bis ( (perfluorohexyl ) sulfonyl) imide . In some embodiments of the method , the fluorous solvent is methoxyperfluorobu tane . In some embodiments of the method , the method further includes eluting the proteins from the reverse phase substrate and analyzing the proteins by mass spectrometry . wherein R is hydrogen , alkyl, substituted alkyl, aryl, Also provided is a composition including a fluorous salt substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, of an ionic liquid and a fluorous solvent. In some embodi substituted heteroalkyl, heteroaryl , substituted heteroaryl, ments of the composition , the fluorous salt of ionic liquid heteroarylalkyl, substituted heteroarylalkyl; includes a fluorinated ion exchanger counterion . In some embodiments of the composition , the fluorinated ion 20 exchanger counterion is described by the formula (VII ) : [ Z ? — ( CH2) m - SO2 - NOSO2 - ( CH2) , Z ? ]. M + ( VII ) where : Z and Z are independently a perfluoroalkyl, an alkyl, a substituted alkyl, a perfluoroaryl, an aryl, or a R - N substituted aryl, wherein Z ' and Z include together a com - 25 bined total of 8 or more fluorinated carbon atoms; m and p are independently 0 , 1 or 2 ; and M + is a cation . In some wherein each of R ' and R² is independently hydrogen , embodiments of the composition , the fluorinated ion alkyl , substituted alkyl, aryl , substituted aryl, arylalkyl , exchanger counterion is bis ( (perfluorohexyl ) sulfonyl) imide . substituted arylalkyl, heteroalkyl, substituted heteroalkyl, In some embodiments of the composition , the fluorous salt 30 heteroaryl , substituted heteroaryl, heteroarylalkyl, substi of an ionic liquid includes a cation ( M + ) selected from the tuted heteroarylalkyl; and group consisting of: f ) Formula (VI ) : a ) Formula (I ) : 35 R ! R -N N R2, R2- N wherein each of Rl and R² is independently hydrogen , wherein each of R1 and R2 is independently hydrogen , alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substi heteroaryl, substituted heteroaryl, heteroarylalkyl , substi tuted heteroarylalkyl. In some embodiments of the compo tuted heteroarylalkyl; 45 sition , the cation is 1 -hexyl - 3 -methyl - imidazolium . In some b ) Formula ( II ) : embodiments of the composition , the fluorous solvent is methoxyperfluorobutane . Also provided is a kit for extracting metabolites from a biological sample . In some embodiments , the kit includes an ionic exchange composition including a fluorinated ion exchanger counterion and a fluorous solvent. In some Z-A embodiments , the kit further includes one or more compo nents selected from the group consisting of: an ionic liquid in an amount sufficient to lyse cells and denature intracel wherein R is hydrogen , alkyl, substituted alkyl, aryl, » lular metabolic enzymes in the biological sample ; a protein substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, and /or lipid adsorbing matrix selected from a reverse phase substituted heteroalkyl, heteroaryl, substituted heteroaryl, matrix , an ion exchange matrix and a size exclusion matrix ; heteroarylalkyl, substituted heteroarylalkyl; and instructions for extracting metabolites from the cells of c ) Formula ( III ): the biological sample . In some embodiments of the kit, the ion exchanger composition is disposed in a plurality of containers . In some embodiments of the kit , the protein and/ or lipid adsorbing matrix is disposed in a plurality of containers . In some embodiments of the kit , the plurality of R 65 containers is a multiwell plate . Also provided is a kit that includes an ionic liquid in an amount sufficient to lyse cells and denature intracellular US 9 ,958 ,363 B2 37 38 metabolic enzymes in the biological sample ; and an ion b ) Formula ( II ) : exchanger composition including a fluorinated ion exchanger counterion . In some embodiments , the kit, further includes one or more components selected from the group consisting of: a fluorous solvent; a protein and /or lipid adsorbing matrix selected from a reverse phase matrix , an ion exchange matrix and a size exclusion matrix ; and Z instructions for extracting metabolites from the cells of the biological sample . In some embodiments of the kit , the ion 10 exchanger composition is disposed in a plurality of contain ers . In some embodiments of the kit, the protein and /or lipid wherein R is hydrogen , alkyl, substituted alkyl, aryl , adsorbing matrix is disposed in a plurality of containers . In substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, some embodiments of the kit , the plurality of containers is substituted heteroalkyl, heteroaryl , substituted heteroaryl, a multiwell plate . * ** 15 heteroarylalkyl, substituted heteroarylalkyl; or Also provided is a system for analytical sample treatment. c ) Formula ( III) : In some embodiments , the system includes a container having disposed therein : an ionic exchange composition including a fluorinated ion exchanger counterion ; and a fluorous solvent. In some embodiments , the system includes 20 a plurality of the containers , each container having disposed R therein the ionic exchange composition and the fluorous solvent. wherein R is hydrogen , alkyl, substituted alkyl, aryl, 25 substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, Alternative Embodiments A substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl; or Further details of some alternative embodiments of the d ) Formula (IV ) : subject method , and systems and kits for performing the same are described below . 30 In some embodiments , the method includes : lysing cells of a biological sample ; and contacting the biological sample Z with an amount of ionic liquid sufficient to denature intra cellular metabolic enzymes in the biological sample to produce a contacted cellular sample . In certain embodiments 35 of themethod , the contacting the biological sample with the ionic liquid lyses the cells of the biological sample . In certain embodiments , the method further includes mixing wherein R is hydrogen , alkyl, substituted alkyl, aryl, the contacted cellular sample with an organic solvent to substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, produce an ionic liquid -organic e solvent composition . InIn 40 substituted heteroalkyl, heteroaryl, substituted heteroaryl, certain embodiments of the method , mixing the contacted heteroarylalkyl, substituted heteroarylalkyl; or cellular sample with the organic solvent includes producing e ) Formula ( V ): a dispersed microdroplet ionic liquid - organic solvent com position . In certain embodiments , the method further includes contacting the ionic liquid -organic solvent compo - 45 sition with an ion exchange composition to produce a second R - NO ionic liquid -organic solvent composition . In certain embodi ments of the method , the ion exchange composition is a second ionic liquid . In certain embodiments of the method , wherein each of R ' and R² is independently hydrogen , the ion exchange composition includes lithium bis [( trifluo - 50 alkyl, substituted alkyl , aryl, substituted aryl, arylalkyl, romethane ) sulfonyl] imide (LiNTf , ) . In certain embodi- substituted arylalkyl , heteroalkyl, substituted heteroalkyl, ments , the method further includes extracting metabolites heteroaryl , substituted heteroaryl, heteroarylalkyl, substi from the ionic liquid . tuted heteroarylalkyl ; or In certain embodiments of the method , the ionic liquid f ) Formula (VI ) : includes a cation selected from the group consisting of: 55 a ) Formula ( I ) :

NO R2 R - NON - R ? , 60 wherein each of R1 and R2 is independently hydrogen , alkyl , substituted alkyl , aryl, substituted aryl, arylalkyl, wherein each of R1 and R² is independently hydrogen , substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkyl, substituted alkyl, aryl, substituted aryl , arylalkyl, heteroaryl , substituted heteroaryl, heteroarylalkyl, substi substituted arylalkyl, heteroalkyl, substituted heteroalkyl, 65 tuted heteroarylalkyl. heteroaryl , substituted heteroaryl, heteroarylalkyl, substi - In certain embodiments of the method , the ionic liquid tuted heteroarylalkyl; or includes 1- butyl - 3- methyl - imidazol- 3- ium . In certain US 9 ,958 ,363 B2 39 embodiments of the method, the biological sample is con tacted with the ionic liquid at room temperature . In certain embodiments , the method further includes filtering the bio logical sample to remove culture media and extracellular GZ+ components from the cells prior to contacting with the ionic 5 liquid . Also provided is a system for high throughput analysis of cellular metabolites. In some embodiments , the system includes : a contacting apparatus configured for contacting 10 wherein R is hydrogen , alkyl, substituted alkyl, aryl, one or more biological samples with an ionic liquid ; a substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl, sampling device configured to provide one or more biologi substituted heteroalkyl, heteroaryl, substituted heteroaryl, cal samples including cells to the contacting apparatus; and heteroarylalkyl, substituted heteroarylalkyl; or e ) Formula an ionic liquid solvent chamber configured to provide one or (V ): more ionic liquids to the contacting apparatus. In certain 15 embodiments , the system further includes an ion exchange composition chamber configured to provide one or more ion exchange compositions to the contacting apparatus . In cer RD - N tain embodiments of the system , the ion exchange compo sition includes lithium bis [( trifluoromethane )sulfonyl ] imide 20 (LiNTf2 ) . In certain embodiments , the system further wherein each of R and R² is independently hydrogen , includes a sample analyzer . In certain embodiments of the alkyl, substituted alkyl , aryl, substituted aryl, arylalkyl, system , the sample analyzer includes liquid - chromatogra substituted arylalkyl, heteroalkyl, substituted heteroalkyl, phy -mass spectrometry or gas chromatography -mass spec heteroaryl, substituted heteroaryl, heteroarylalkyl, substi trometry . In certain embodiments of the system , the ionic 25 tuted heteroarylalkyl ; or f) Formula (VI ) : liquid includes a cation selected from the group consisting of: a ) Formula ( I) :

30 R2 R ! - NON - R2, wherein each of R and R2 is independently hydrogen , alkyl, substituted alkyl, aryl , substituted aryl, arylalkyl, wherein each of R1 and R2 is independently hydrogenon , 35 substituted arylalkyl, heteroalkyl, substituted heteroalkyl, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substi substituted arylalkyl, heteroalkyl, substituted heteroalkyl, tuted heteroarylalkyl. heteroaryl, substituted heteroaryl, heteroarylalkyl , substi In certain embodiments , the system further includes a tuted heteroarylalkyl; or b ) Formula (II ): processor including memory operably coupled to the pro 40 cessor, wherein the memory includes instructions stored thereon , the instructions including : algorithm for contacting one or more biological samples with an amount of ionic liquid sufficient to lyse cells and denature intracellular metabolic enzymes in the biological sample ; algorithm for 45 mixing the ionic liquid with an organic solvent to produce a OZ first ionic liquid -organic solvent two - phase composition ; algorithm for employing an agitator to agitate the first ionic liquid - organic solvent two - phase composition to produce a dispersed microdroplet composition ; algorithm for contact wherein R is hydrogen , alkyl, substituted alkyl, aryl, 30 ing dispersed microdroplet composition with an ion substituted aryl, arylalkyl , substituted arylalkyl, heteroalkyl, exchange composition to produce a second ionic liquid substituted heteroalkyl , heteroaryl, substituted heteroaryl, organic solvent two - phase composition ; algorithm for sepa heteroarylalkyl, substituted heteroarylalkyl; or c ) Formula rating the ionic liquid from the organic solvent and extract ( III) : ing metabolites from the ionic liquid ; and instructions for identifying one or more metabolites extracted from the ionic liquid . Also provided is a kit for extracting metabolites from a biological sample . In some embodiments , the kit includes : 60 one or more ionic liquids in an amount sufficient to lyse cells R and denature intracellular metabolic enzymes in the biologi cal sample ; one or more organic solvents; one or more ion wherein R is hydrogen , alkyl, substituted alkyl, aryl, exchange compositions ; and instructions for extracting substituted aryl, arylalkyl, substituted arylalkyl , heteroalkyl, metabolites from the cells of the biological sample . substituted heteroalkyl, heteroaryl, substituted heteroaryl, 65 Further details of some implementation of the methods, heteroarylalkyl, substituted heteroarylalkyl; or d) Formula and some components of the systems and kits of the alter (IV ) : native embodiments described above may be described in US 9 ,958 ,363 B2 41 specification and figures of U . S . application Ser. No . 14 /205 , In some embodiments , the method comprises : ( a ) extract 100 , filed Mar. 11, 2014 , which application is incorporated ing an aqueous sample comprising an ionic liquid with a herein by reference . perfluorous ion exchanger phase comprising a perfluorous counterion to produce an extracted aqueous sample , wherein Alternative Embodiments B 5 the perfluorous counterion and the ionic liquid form a perfluorous salt that is soluble in the perfluorous ion Further alternative embodiments of the subject method exchanger phase; ( b ) passing the extracted aqueous solution are described below . through a fluorous affinity chromatography column under In some embodiments , themethod is a method for remov conditions in which the perfluorous salt, if present, is ing an ionic liquid from an aqueous sample . In some 10 absorbed to the column ; and ( c ) collecting the eluate of the embodiments , the method comprises: ( a ) combining an extracted aqueous solution . In certain embodiments of the aqueous sample comprising an ionic liquid with a perfluo - method , the aqueous sample comprises cellular metabolites rous counterion to produce an aqueous solution comprising and the method further comprises analyzing the aqueous a perfluorous salt of the ionic liquid ; ( b ) contacting the solution using mass spectroscopy . aqueous solution with a fluorous affinity substrate , thereby In some embodiments , the method comprises: ( a ) com absorbing the perfluorous salt on the fluorous affinity sub - bining an aqueous sample comprising an ionic liquid with an strate ; and ( c ) collecting the eluate of step ( b ) . In certain ion exchanger counterion to produce an aqueous solution embodiments , the method further comprises , prior to step comprising a perfluorous salt of the ionic liquid , wherein at ( b ) , extracting a portion of the perfluorous salt of the ionic 20 least one of the ionic liquid and the ion exchanger counterion liquid from the aqueous sample using a perfluorous ion is fluorinated ; ( b ) contacting the aqueous solution with a exchanger solution that is immiscible with the aqueous fluorous affinity substrate , thereby absorbing the perfluorous sample , wherein the perfluorous ion exchanger solution salt on the fluorous affinity substrate ; and ( c ) collecting the comprises the perfluorous counterion . In certain embodi- eluate of step ( b ) . In certain embodiments of the method , the ments of the method , the aqueous sample comprises cellular 25 ionic liquid is fluorinated . In certain embodiments of the metabolites . In certain embodiments , the method further method , the ion exchanger counterion is a perfluorous coun comprises analyzing the eluate using mass spectroscopy . terion . In certain embodiments of the method , the aqueous In certain embodiments of the method , the perfluorous sample comprises cellular metabolites and the method fur counterion is described by formula (VII ) : Z - L - A - ( -L2 - Z ) . ther comprises analyzing the eluate using mass spectros (VII ) wherein : Z is a perfluoroalkyl, a perfluoroaryl , a 30 copy. fluorinated alkyl or a fluorinated aryl; n is 0 , 1 , 2 or 3 ; each In some embodiments , the method may involve a photo Z ? , if present, is independently selected from the group cleavable ionic liquid . In such cases, removal of the photo consisting of a perfluoroalkyl, a perfluoroaryl, an alkyl, a cleavable ionic liquid from a sample of interest may be substituted alkyl , an aryl and a substituted aryl; A is a facilitated by application of light to photocleave the ionic charged moiety capable of acting as a counterion to the ionic 35 liquid , see e . g ., FIG . 10 . Cleavage of the ionic liquid may liquid ; andLand L are independently a covalent bond or provide two or more fragments, where some of the frag a linker. In certain embodiments of the method , the perfluo ments produced are more easily extracted or separated from rous counterion is described by formula (VIII ) : Z ' - L - A the sample of interest using the subject methods. In some ( VIII ) wherein Z ' , L ' and A are as defined above . In certain cases, photocleavage of the ionic liquid produces fragments embodiments of the method , A is an organic anion selected 40 that provide for an improved analysis by mass spectroscopy from a sulfonimidate , a sulfonate , a carboxylate , a phosphate because the fragments are either : more easily removed from and a borate . In certain embodiments of the method , when the analytical sample ; or cause less interference during MS n = 1 , Ais — SO , NOSO , — . In certain embodiments of the analysis and thus have less need to be removed prior to method , when n = 0 , A is SO NHO ) . In certain embodi- analysis . ments of the method , L and each L ? are independently a 45 In some embodiments , the method may involve use of a C . -C . alkyl linker or a covalent bond . In certain embodi- bioorthogonal conjugation chemistry to remove the ionic ments of the method , L ' and each L are independently liquid from a sample of interest via the formation of a selected from CH2CH2— or a covalent bond . In certain covalent bond to a support including a compatible functional embodiments of the method , Z and Z2 are each indepen - group . In some embodiments, the ionic liquid includes a first dently a perfluoroalkyl group ( e . g . , comprising 4 or more 50 functional group capable of conjugation to a second com fluorinated carbon atoms) . In certain embodiments of the patible functional group . Any convenient conjugation chem method , Z ' and each Z² comprise together a combined total istries may be utilized . FIG . 11 illustrates one embodiment, of 8 or more fluorinated carbon atoms ( e. g ., 10 or more , 12 where an azide - linked ionic liquid that may be conjugated to or more , 14 or more, 16 or more, 18 or more or even 20 or an alkyne - solid support ( e . g ., cyclooctyne -solid support) via more fluorinated carbon atoms) . In certain embodiments of 55 Click chemistry to produce an immobilized ionic liquid the method , the perfluorous counterion is described by the which may be subsequently removed from a sample of formula (IX ) : [Z1 - ( CH , ) . S0 , - NOSO , — (CH , ) . — interest . In other embodiments , an alkyne - linked ionic liquid Z ? ]. M + ( IX ), wherein : Z and Z ? are independently a per - is conjugated to an azide - solid support via Click chemistry fluoroalkyl, an alkyl, a substituted alkyl, a perfluoroaryl, an to produce an immobilized ionic liquid which may be aryl, or a substituted aryl, wherein Z and Z - comprise 60 subsequently removed from a sample of interest. together a combined total of 8 or more fluorinated carbon Also provided are systems and kits for practicing the atoms ( e . g . , 10 or more , 12 or more , 14 or more , 16 or more , embodiments of the method . 18 or more or even 20 or more fluorinated carbon atomsm ) ; F urther details of some implementation of the alternative and p are independently 0 , 1 or 2 ; and M + is a cation ( e . g . , embodiments of the method and components of systems and Li+ , K + , etc ) . In certain embodiments of the method , the 65 kits for practicing the same are described above may be perfluorous counterion is a bisnonafluoro - 1 -butanesulfon - described in specifications and figures of U . S . provisional imidate . application Ser . No. 62 /016 , 003 , filed on Jun . 23 , 2014 ; and US 9 , 958 , 363 B2 43 44 U .S . provisional application Ser . No . 62 /051 , 804 , filed on Example 1 Sep . 17 , 2014 , which applications are incorporated herein by reference . One embodiment of the method utilizes a fluorous oil (HFE -7100 , methoxyperfluorobutane ) and a silver salt of Alternative Embodiments C 5 bis (( perfluorohexyl ) sulfonyl) imide , which is very water insoluble , along with multiple extractions as a way to Further alternative embodiments of the subject method remove hexylmethylimidazolium chloride from an aqueous are described below . metabolite solution . The HFE - 7100 solubilizes the silver ( 1 ) In some embodiments , the method is a method for pro bis ( ( perfluorohexyl) sulfonyl ) imide just enough so that as the cessing a sample including : ( a ) combining an aqueous 10 ion exchange reaction starts to occur, the new ionic liquid sample including an ionic liquid with a magnetic ionic that is formed helps solubilize the remaining silver( I ) bis exchanger to produce an aqueous solution including a mag ( (perfluorohexyl ) sulfonyl) imide . As a byproduct, the AgCl that is formed is very insoluble in aqueous solutions and netic salt of the ionic liquid ; ( b ) applying an external precipitates as a new solid . This is important to keep it out magnetic field to the product of step ( a ) to remove the 15 of the mass spectrometer for analysis purposes and for magnetic salt from the sample ; and ( c ) collecting the sample . cleanliness of the mass spectrometer . In certain embodiments of the method , the combining step One workflow of interest is depicted in FIGS . 1 and 2 . In ( a ) results in a biphasic liquid . In certain embodiments of the summary the cells are lysed and metabolism is quenched by method , the aqueous sample includes cellular metabolites . In adding greater than 30 % aqueous hexamethylimidazolium certain embodiments , the method further includes analyzing 20 chloride ( typically 50 % ) and vortexing , followed by passage the eluate using mass spectroscopy. In certain embodiments through a C18 spin column to remove proteins, which keeps of the method , the magnetic ionic exchanger includes a metabolism quenched . Next, the solution is added to a vial transition metal. In certain embodiments of the method , the containing a fluorous ion exchanger ( silver( I ) ionic salt is transition metal complex susceptible to an bis ( ( perfluorohexyl )sulfonyl ) imide ) that is present as the external magnetic field . In certain embodiments of the 25 silver ( 1 ) salt as a solid in a fluorous solvent (specifically method , the magnetic ionic exchanger includes a magnetic HFE -7100 , which is methoxyperfluorobutane) . The mixture counterion capable of forming a salt with the ionic liquid , is vortexed , spun in a centrifuge , and the aqueous layer is e . g ., imidazolium , that has limited or no solubility in water. transferred to a new vial containing a small amount of the In certain embodiments of the method , the magnetic ionic silver ( I ) bis ( ( perfluorohexyl) sulfonyl ) imide in HFE -7100 to exchanger includes a paramagnetic or ferromagnetic ele - 30 do another extraction . This process may be repeated four ment. In certain embodiments of the method , the magnetic times and the ionic exchanger includes a CoCl2 or FeCl2 counterion . aqueous layer is then ready for injection into the MS (mass Further details of some implementation of the alternative spectrometer ) . Optionally , fluorous solid phase extraction embodiments of the method described above may be can be performed if necessary to remove all traces of the described in specification and figures of U . S . provisional 35 silver ( I) bis (( perfluorohexyl ) sulfonyl) imide . application Ser. No . 62 /049 , 285 , filed on Sep . 11, 2014WA , FIG . 4 depicts microscope images demonstrating lysis of which application is incorporated herein by reference . yeast cells that have been treated with water ( left ) or 1 : 1 water /HMIM -C1 ( right ) and stained with trypan blue . Alternative Embodiments D FIG . 5 illustrates the results of a ATP Luciferase assay that 40 demonstrates ATP metabolism in E . coli cells remains Further alternative embodiments of the subject method quenched in aqueous HMIM - Cl solutions . are described below . FIG . 6 illustrates the removal of proteins using a C18 SPE In certain cases , the method may involve : a ) mixing a cell ( solid phase extraction ) prior to ion exchange reaction keeps with an ionic liquid ( e . g . , an ionic liquid /water solution ) , metabolism quenched . thereby quenching metabolism and lysing the cells , and b ) 45 FIG . 7 shows extracted ion chromatograms ( EIC ) for performing reverse phase chromatography on the product of HMIM after each extraction of the aqueous layer using fresh step a ) ( e .g ., using a C18 column ) , thereby removing the HFE -7100 (methoxyperfluorobutane ) and bis ( (perfluoro protein from the product of step a ). These steps do not utilize hexyl) sulfonyl) imide . ( A ) EIC for HMIM in a blank run , an organic solvent. In some embodiments , this method may after the initial ion exchange reaction , and two subsequent further include : c ) adding an aqueous exchange solution 50 extractions. ( B ) The same EICs as in ( A ) , with the initial ion ( LiNTf, ) to the flow through of step b ) , thereby producing exchange reaction EIC removed to demonstrate the levels go a first aqueous phase that contains metabolites and a second almost back to background. hydrophobic phase containing the ionic liquid . In some embodiments , this method may further include : d ) analyzing Example 2 the metabololites in the aqueous phase , e . g . , by LC -MS . An 55 example of the workflow is shown in FIG . 9 . Sample processing and preparation for metabolomic Further details of some implementation of the alternative analysis which incorporates into its steps one or more of the embodiments of the method described above may be following : described in the specification and figures of U . S . provisional 1 . Quenching — Stopping any metabolic processes such application Ser. No . 62/ 016 , 000 , filed on Jun . 23 , 2014 , 60 that an accurate snapshot of the current metabolic state which application is incorporated herein by reference . of the cells under study can be evaluated . 2 . Cell Lysis — The intracellular metabolome is measured . EXAMPLES The cell is lysed and the cell contents separated from the extracellular medium . The following example is offered for illustrative purposes 65 3 . Metabolite Extraction : Metabolic components are only , and is not intended to limit the scope of the present extracted from all other cellular components ( proteins, invention in any way . nucleic acids, lipids ) . US 9 ,958 ,363 B2 45 46 4 . Metabolite Concentration : Depending on the sensitivity Step . 6 : The Ionic liquid is removed from the metabolite of the analytical technique and the requirements of the analytes by solid phase microextraction under conditions experiment, the extracted metabolites are concentrated where the ionic liquid itself does not act as an elutropic prior to analysis . solvent during the loading of analytes onto stationary phase . 5 In the above example , ionic liquids of interest have the Brief Description of Exemplary Techniques following characteristics: ( a ) Dispersive Liquid Liquid Microextraction (DLLME ) , Denature Proteins/ Quench Metabolites is a sample preparation technique based on formation of a Lyse Cells (Yeast ) turbid solution by quickly injecting a mixture of an extrac - 10 Extract/ Solublize Metabolites of interest tion solvent and a disperser solvent into an aqueous solution . Precipitate /Remove Proteins The extraction solvent is hydrophobic and of higher density Immiscible w /Organic for 2 phase extraction DLLME than water while the disperser is miscible with both aqueous Metabolites can be separated by liquid chromatography and organic phases . The obtained turbid solution results in (e . g ., HPLC ) the large contact area between the fine extraction solvent 155 In one example , the ionic liquid include: droplets and aqueous analyte solution , remarkably decreas ing the extraction time and increasing the extraction effi H3C ciency . DLLME has been widely applied to arrange of analytical samples , primarily environmental. Ny BMIM + NEN ( b ) Ionic Liquids combined with DLLME for preparing a 2420 sample where the compounds of interest are extracted into N ( CN ) 2 the ionic liquid . A method for metabolic sample preparation based on Dispersive Liquid Liquid Microextraction (DLLME ) utiliz - 25 CH2 ing Ionic Liquids ( IL ) in which the novel ionic liquids rapidly and effectively denature metabolic enzymes to Alternatively , room temperature ionic liquids may include quench metabolism and simultaneously extracting hydro - a scaffold structure such as a 1 , 3 substituted imidazolium philic metabolites in the background of cellular components . cation , a salt can be structured , by varying the R groups and Ionic Liquids are used in combination with DLLME to 30 the counter ion to optimize a range of physicochemical simultaneously lyse cells and denature metabolic enzymes in properties . such a way as to rapidly quench metabolism . The workflow may have at least the following benefits : 1 . Rapid Cell Lysis and Metabolic Quenching without the use of cryo -conditions . 3535 REAR 2 . Rapid separation of extra - and intra -cellular compo nents 3 . Rapid and Efficient fractionation of intracellular com 1 , 3 substituted imidazol- 3 - ium ponents into hydrophilic and hydrophobic fractions. 4 . Robust technique applied to a range of cellular systems 40 For example , in the structure show above , R and R2 are without the need of optimization for each cell type selected and adapted based on general physico - chemical 5 . Easily adapted to automated , robotic platforms and properties such as density , solubility , vapor pressure , as multiwell plate sample formats . desired . In addition , specific substitutions can be made on the R - groups to enhance specific chemical interactions with Example 3 45 target groups. An example workflow includes : Example 4 Step 1 : Cell Suspension is transferred to a filter tube and rapidly filtered to separate culture media and extra - cellular so . An example workflow similar to the steps described components from the cell mass. above in Example 2 include a three phase system in which Step 2 : The filtered cells are resuspended in a hydrophilic an ionic liquid is used that can interact with metabolites to Ionic Liquid . This simultaneously and rapidly lyses the cells extract them from aqueous solution intermediate between and denatures the metabolic enzymes and consequently the organic solution . quenches metabolic processes. 55 Step 1: Cell Suspension is transferred to a filter tube and Step 3 : The Ionic Liquid containing the sample is mixed rapidly filtered to separate culture media and extra -cellular with a hydrophobic organic liquid . components from the cell mass . Step 4 : The two phase system is agitated forming a dispersed Step 2 : The filtered cells are resuspended as an aqueous system of ionic liquid microdroplets with a high surface area solution in a hydrophilic Ionic Liquid . This would simulta (without the addition of a dispersant) . Hydrophobic compo - 60 neously rapidly lyse the cells and denature the metabolic nents ( such as lipids ) are extracted into the organic phase . enzymes and consequently quench metabolic processes. Proteins precipitate . The hydrophilic metabolite analytes are Step 3 : The Ionic Liquid containing the sample is mixed extracted into the ionic liquid . with a hydrophobic organic liquid . Step 5 : An Ion Exchanger Composition ( e . g ., LinTf, ) is Step 4 : The three phase system is agitated forming a added causing the Ionic Liquid microdroplets to condense in 65 dispersed system of ionic liquid microdroplets with a high a salt metathesis reaction and separate from the organic surface area (without the addition of a dispersant) . Hydro layer . phobic components ( such as lipids) are extracted into the US 9 ,958 ,363 B2 47 48 organic phase . Proteins precipitate . The hydrophilic metabo - from the solution to produce the aqueous eluate, wherein the lite analytes are extracted into the ionic liquid . fluorous solvent is immiscible with the solution . Step 5 : An Ion Exchanger ( e . g ., LiNTf2 ) is added causing the Ionic Liquid microdroplets to condense in metathesis reac 3 . The method of claim 1 , wherein the fluorous affinity tion and separate from the organic and aqueous layers. material is a fluorous affinity chromatography support that Step . 6 : The Ionic liquid is removed from the metabolite adsorbs the fluorous salt from the solution to produce the analytes by solid phase microextraction under conditions aqueous eluate . where the ionic liquid itself does not act as an elutropic 4 . The method of claim 1 , wherein the ionic liquid solvent during the loading of analytes onto stationary phase . comprises a cation selected from the group consisting of: Example 5 10 a ) Formula ( I) : An example workflow similar to the steps described above in Example 2 , includes a three phase system in which R ! - NON - R2, an ionic liquid is used that can interact with metabolites to 15 extract them from aqueous solution intermediate between the organic solution with the addition of a chaotrope and dispersant to enhance microdroplet formation . wherein each of Rl and R2 is independently hydrogen , Although the foregoing invention has been described in alkyl, substituted alkyl , aryl, substituted aryl, arylalkyl, some detail by way of illustration and example for purposes substituted arylalkyl, heteroalkyl, substituted het of clarity of understanding , it is readily apparent to those of eroalkyl, heteroaryl, substituted heteroaryl, heteroaryl ordinary skill in the art in light of the teachings of this alkyl, substituted heteroarylalkyl; invention that certain changes and modifications may be b ) Formula ( II ): made thereto without departing from the spirit or scope of 25 the appended claims. Accordingly , the preceding merely illustrates the prin ciples of the invention . It will be appreciated that those skilled in the art will be able to devise various arrangements which , although not explicitly described or shown herein , 30 OZ embody the principles of the invention and are included within its spirit and scope . Furthermore , all examples and conditional language recited herein are principally intended wherein R is hydrogen , alkyl, substituted alkyl, aryl, to aid the reader in understanding the principles of the substituted aryl, arylalkyl, substituted arylalkyl, het invention and the concepts contributed by the inventors to 35 eroalkyl, substituted heteroalkyl , heteroaryl, substi furthering the art, and are to be construed as being without tuted heteroaryl, heteroarylalkyl, substituted heteroary limitation to such specifically recited examples and condi lalkyl ; tions . Moreover, all statements herein reciting principles, aspects , and embodiments of the invention as well as spe c ) Formula (III ) : cific examples thereof, are intended to encompass both * structural and functional equivalents thereof. Additionally , it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i. e . , any elements developed that perform the same function , 45 regardless of structure . The scope of the present invention , therefore , is not intended to be limited to the embodiments shown and described herein . Rather, the scope and spirit of wherein R is hydrogen , alkyl, substituted alkyl, aryl, present invention is embodied by the appended claims. substituted aryl , arylalkyl, substituted arylalkyl, het What is claimed is : 5050 eroalkyl, substituted heteroalkyl , heteroaryl, substi 1 . A method for removing an ionic liquid from an aqueous tuted heteroaryl, heteroarylalkyl, substituted heteroary sample comprising a cell lysate , the method comprising : lalkyl; ( a ) combining an aqueous sample comprising a cell lysate and an ionic liquid with an ion exchanger composition comprising an ion exchanger counterion , thereby pro - 55 ducing a solution comprising the cell lysate and a -Z fluorous salt of the ionic liquid , wherein at least one of the ionic liquid and the ion exchanger counterion is fluorinated ; (b ) contacting the solution with a fluorous affinity mate - 60 rial , thereby removing the fluorous salt of the ionic liquid from the solution and producing an aqueous d ) Formula (IV ): eluate comprising the cell lysate ; and wherein R is hydrogen , alkyl, substituted alkyl, aryl, ( c ) collecting the aqueous eluate comprising the cell substituted aryl, arylalkyl, substituted arylalkyl, het lysate . eroalkyl, substituted heteroalkyl, heteroaryl, substi 2 . The method of claim 1, wherein the fluorous affinity tuted heteroaryl, heteroarylalkyl, substituted heteroary material is a fluorous solvent that extracts the fluorous salt lalkyl; US 9 , 958 , 363 B2 49 50 e ) Formula ( V ): 6 . The method of claim 1, wherein the ionic liquid comprises 1 -hexyl - 3 -methyl - imidazolium and the ion exchanger counterion is bisnonafluoro - 1 - butanesulfonimi date or bis ( (perfluorohexyl ) sulfonyl) imide . 7 . The method of claim 1 , wherein the ion exchanger R - NS composition comprises a salt of the ion exchanger counte rion selected from silver, lithium , sodium and potassium . wherein each of R is hydrogen , alkyl, substituted alkyl, 8 . The method of claim 1 , wherein the ion exchanger aryl, substituted aryl, arylalkyl, substituted arylalkyl, composition comprises a silver salt of the ion exchanger heteroalkyl, substituted heteroalkyl, heteroaryl, substi - 10 counterioninsoluble silverand saltstep . (a ) further comprises producing an tuted heteroaryl, heteroarylalkyl, substituted heteroary 9 . The method according to claim 1 , further comprising, lalkyl; and prior to step ( a ), contacting the aqueous sample comprising f ) Formula (VI ) : the ionic liquid with a reverse phase substrate , thereby adsorbing proteins and /or lipids on the reverse phase sub 15 strate , if present in the aqueous sample . 10 . The method according to claim 1 , further comprising analyzing the aqueous eluate by mass spectrometry 11 . The method according to claim 1 , further comprising : lysing cells of a biological sample ; and wherein each of R1 and R² is independently hydrogen , 20 contacting a biological sample with an amount of the alkyl, substituted alkyl , aryl, substituted aryl, arylalkyl, ionic liquid sufficient to denature intracellular meta substituted arylalkyl, heteroalkyl , substituted het bolic enzymes in the biological sample to produce the eroalkyl, heteroaryl, substituted heteroaryl, heteroaryl aqueous sample . alkyl, substituted heteroarylalkyl. 12 . The method of claim 11 , wherein the ionic liquid 5 . The method of claim 1 , wherein the ion exchanger 2523 composition is an aqueous composition comprising 30 % or counterion is a fluorinated counterion described by the more of the ionic liquid . formula (VII ) : 13 . The method of claim 5 , wherein the ionic liquid comprises 1 -hexyl - 3 -methyl - imidazolium and the fluori [ Z _ CH2) m - S02 — NO — S02 (CH2 ) p — Z2 ]. M + (VII ) nated ion exchanger counterion is bis ( (perfluorohexyl ) sul wherein : 30 fonyl) imide . Z ' and Z are independently a perfluoroalkyl, an alkyl, a 14 . The method of claim 2 , wherein the fluorous solvent substituted alkyl, a perfluoroaryl, an aryl, or a substi is methoxyperfluorobutane. tuted aryl, wherein Z ' and Z2 comprise together a 15 . The method of claim 9, further comprising eluting the combined total of 8 or more fluorinated carbon atoms; 35 proteins from the reverse phase substrate and analyzing the m and p are independently 0 , 1 or 2 ; and proteins by mass spectrometry . M + is a cation . * * * * *