Sample Preparation. Method Development

Transcription

1 Sample Preparaton Method Development Wherever you see ths symbol, t s mportant to access the on-lne course as there s nteractve materal that cannot be fully shown n ths reference manual.

2 Ams and Objectves Ams and Objectves Ams To nvestgate varous aspects of practcal SPE ncludng: o Sorbent Column Processng o Sorbent Bed Geometry o The use of Dsk Based SPE o The mpact of fnes on SPE To descrbe the basc prncples and stages of method development n SPE To gve an overvew of troubleshootng SPE protocols and gve specfc advce for varous problems that are routnely encountered To descrbe the basc prncples of Generc Methods n SPE Objectves At the end of ths Secton you should be able to: Identfy the optmum condtons for processng your SPE protocol ncludng varous advanced concepts such as Sorbent Soak Steps Evaluate the sorbent mass and geometry most approprate for your method Approach SPE method development n a logcal fashon and optmse your SPE protocol for maxmum senstvty and specfcty Undertake troubleshootng procedures for varous common protocol ssues Identfy when the use of generc SPE methods mght be approprate and descrbe how mxed mode SPE mght be used n ths stuaton

3 Content Sorbent Column Processng 3 Centrfugal Column Processng 4 Vacuum Column Processng 5 Dsadvantages of Vacuum Column Processng 6 Sorbent Column Processng 7 Sorbent Bed Geometry 8 Dsk Products for SPE 9 Dsk Based Sorbents 12 Fnes 13 Fnes Channellng 15 SPE Method Development Overvew 16 Analyte Assessment I 18 Analyte Assessment II 19 Analyte Assessment III 21 Mechansm Selecton 23 Mechansm Selecton 25 Sorbent Screenng Alternatve Sorbents I 28 Sorbent Screenng Alternatve Sorbents II 30 Sorbent Screenng Process 31 Procedure Optmzaton I 35 Procedure Optmzaton II 36 Procedure Optmzaton III 38 Soak Steps 39 Dryng Steps 42 Eluton Optmsaton 43 Eluton Optmsaton Results 44 SPE Method Troubleshootng Overvew 45 Recovery Problems I 46 Recovery Problems II 48 Recovery Problems III 51 Reproducblty Problems I 55 Reproducblty Problems II 56 Cleanlness Problems 57 Cleanlness Problems Alternatve Solvents 59 Cleanlness Problems Poor Sorbent Selectvty 60 Inadequate Throughput 62 Generc Methods n SPE Overvew 66 Mxed Mode Sorbents 68 Generc Methods Overvew 69 Generc Methods Basc Protocol 70 Generc Methods Further Optmzaton 71 Crawford Scentfc 2

4 Sorbent Column Processng Most SPE columns contatn partcles of a suffcently small sze such that consstent flow through the column requres some motve force; that s, does not occur through gravty alone. Although there are commercal exceptons to ths for some steps (partcularly wth fnes-free sorbents), the sample applcaton step for most pharmaceutcal samples often nvolves somewhat vscous bologcal fluds, and these samples are best asssted n movng through the column. There are three prmary approaches to facltatng solvent or sample processng through SPE columns: a) Centrfugal force b) Vacuum c) Postve pressure Solvent or sample processng through SPE columns Crawford Scentfc 3

5 Centrfugal Column Processng Centrfugal force nvolves the use of specfc commercal centrfuges that offer avalable baskets for SPE column processng. Wth the use of these baskets, many SPE columns may often be processed at one tme. The advantages of centrfugal processng are: 1) Large numbers of columns processed smultaneously. 2) Very precse control of solvent or sample flow rates through the columns. Dsadvantages nclude: 1) Long acceleraton and deceleraton tmes (ths may be offset by the ablty to process many samples smultaneously) 2) Inconvenent access to the columns to add solvents and samples 3) Inablty to observe the flow process through the column. In general, the best applcaton of centrfuges for sample processng s for very large numbers of samples, where adequate tme may be spent n optmzng a sngle procedure to be used over and over agan. SPE tubes processed usng centrfugal force Crawford Scentfc 4

6 Vacuum Column Processng The most common approach to SPE column processng nvolves the use of vacuum manfolds. These devces work by nsertng the sorbent column nto the top of the manfolds, to whch a vacuum s appled, most commonly wth a water asprator, nexpensve vacuum pump, or house vacuum. As the pressure nsde the manfold s reduced, the atmospherc pressure above the column forces the sample or solvent through the column bed. Collecton racks may be placed wthn the manfold to collect column effluents, or the racks may be left out and the effluent sent to a waste trap f collecton s unnecessary. Advantages of manfold processng nclude: 1) Ready access to the top of the extracton column to add protocol solvents for each step 2) Ready avalablty of vacuum sources va house vacuum or low-cost vacuum pumps 3) Relatvely low cost of the processng manfolds 4) Ablty to observe the extracton process vsually 1 2 SPE tubes processed usng a vacuum manfold wth tube rack for eluate collecton Crawford Scentfc 5

7 Dsadvantages of Vacuum Column Processng The major dsadvantages of vacuum processng are: 1) Lmtaton of processng pressure to atmospherc pressure, 2) Varablty n column-to-column flow rates. Dfferences n column flow rates may be due to ether slght varatons n the column packed beds from the manufacturer, or dfferences n sample vscosty. These varatons can cause rreproducble results f methods are not extremely rugged. One partcularly nsdous problem s that durng processng, once a sngle or multple columns have run dry, the vacuum dfferental for the rest of the columns s reduced, slowng the flow down and sometmes stoppng t altogether. Nevertheless, vacuum processng contnues to be the most popular approach. Varablty n column to column flow may cause vacuum changes leadng to unequal protocol solvent flow through varous SPE columns Crawford Scentfc 6

8 Sorbent Column Processng The thrd approach to SPE column processng, whch s growng n popularty, s the use of pressure manfolds. These devces hold the columns n place on a platform, so that an upper manfold applyng postve pressure may be nterfaced to the columns va a smple gasket seal. The pressure supply may be house or tank-based ntrogen or ar. The pressure manfold typcally uses fxed orfce nozzles, whch provde very unform pressure to all of the columns, regardless of backpressure. Therefore, even f all of the columns except one have run dry, the remanng column stll receves adequate pressure for solvent flow. Thus the advantages of pressure processng nclude: 1) Unformty of column processng. 2) Relatvely low cost. 3) Ablty to vsually observe the extracton process. The prmary dsadvantages of pressure processng are: 1) Successve protocol solvents cannot be added to the columns as readly as wth vacuum processng. 2) The potental for sample-to-sample cross-contamnaton va the sealng gasket f the user s not fastdous n sample loadng. SPE tubes processed usng postve pressure manfold Crawford Scentfc 7

9 Sorbent Bed Geometry Sorbent bed geometry refers to the relatve wdth of a sorbent bed versus the heght of the bed. Ths s an mportant parameter n SPE columns and can affect the reproducblty of extracton methods. In general, t s recommended to use the wdest bed avalable for a gven mass of sorbent. The justfcaton for ths s as follows: Column flow rate s a partcularly mportant parameter n SPE. In partcular, the flow rate through the columns should not be too fast, or analyte breakthrough wll result. Flow rate s a readly observable phenomenon, and therefore s the most convenent reference for solvent flow. However, the actual parameter that s crtcal s not the flow rate but the lnear velocty through the sorbent bed. Lnear velocty refers to the speed wth whch solvent flows through any partcular pont across the surface of the bed at any tme. Analyte breakthrough results when lnear veloctes across the bed are too hgh. Lnear velocty on two dfferent SPE columns For a gven flow rate, the lnear velocty across the bed s a functon of both the flow rate and the horzontal cross-sectonal area of the bed. For a very narrow bed of a gven sorbent mass, the lnear velocty may be qute hgh for any gven flow rate. The same mass contaned n a wder bed wll exhbt a much slower lnear velocty for the same flow rate, snce the flow s dstrbuted across the surface of the bed. Thus, the use of wder bed geometres for a gven mass of sorbent wll reduce the lnear velocty, and help to ensure a suffcently slow flow through the column to prevent analyte breakthrough. One corollary to ths s that wth a wder bed, faster flow rates may be used to generate the same lnear velocty. If large sample volumes are to be processed, the use of wder beds can therefore accommodate faster sample processng. Crawford Scentfc 8

10 1 2 Dsk Products for SPE Keepng constant lnear velocty on two dfferent SPE columns The orgnal products developed commercally for SPE nvolved bulk partculate sorbents packed n columnar formats such as syrnge barrels, sometmes referred to as powder columns. More recently, an addtonal category of SPE products has been developed, wth much wder bed formats and much shorter bed heghts. These products are typcally referred to as dsk products for SPE. 1 2 Dsk products for SPE Crawford Scentfc 9

11 Dsk products employ two prmary advantages of short, wde packed beds: 1) Much lower lnear veloctes across the bed for a gven flow rate, as compared to longer, narrower beds. 2) Much lower backpressures across the sorbent bed, due to the short path length. Lower backpressures have an addtonal postve corollary, whch s the ablty to use smaller partcle sorbents n the columns (whch nduce hgher backpressure), yet stll allow processng usng conventonal SPE approaches, such as vacuum, postve pressure, and centrfugal force. 1 2 Packed beds One of the ssues facng dsk manufacturers s the challenge of provdng a very short chromatographc bed path wthout the problem of channelng. Channels are free flow paths through the sorbent bed, where an analyte molecule may pass wthout encounterng the chromatographc medum. As n hgh-performance chromatography, channeled columns exhbt excessve band spreadng and neffcent extracton, leadng to analyte breakthrough. There are three prmary commercal approaches (all patented by specfc manufacturers) to creaton of dsk format beds wthout channels, each used by one or more of the man manufacturers of dsk products. Crawford Scentfc 10

12 Channels through the sorbent bed Crawford Scentfc 11

13 Dsk Based Sorbents The frst approach to dsk formats s to embed sorbent partcles wthn a fbrous matrx, smlar to a fabrc. The fabrc-lke matrx, most commonly ether Teflon or glass fbers, provdes a structure whch contans the sorbent partcles, yet stll allows lqud flow to pass. Empore dsks A second approach, whch begns wth glass fbers, s to grow the functonal group chemstry drectly onto the fbers. Ths approach has the advantage over the prevous approach of provdng a more free-flowng devce wth fewer pluggng problems. Fbre dsks The thrd approach s to pack mcro-partculate sorbents usng a slurry technque, whch obvates the need for an entranng fabrc or fbrous matrx. The product, sometmes referred to as a powder dsk, offers the flexblty n sorbent chemstry choces of conventonal powder columns, yet carres the performance benefts of dsks, ncludng hgh-effcency extracton and low lnear veloctes. Crawford Scentfc 12

14 SPE dsks Fnes The term fnes as appled to SPE refers to sorbent partcles that are much smaller n sze than the mean partcle sze specfed by the commercal suppler of an SPE product. Fnes are of mportance due to the many negatve effects they can have on the SPE process and the qualty of the fnal extract. Crawford Scentfc 13

15 Fnes orgnate n the abrason of sorbent partcles aganst one another, and are especally a problem for rregular slcas. As the unbonded slca partcles rub aganst one another durng ntal shppng and handlng, sharp corners and abutments are broken off by neghborng partcles, n much the same way a rock tumbler wll break off corners and edges of the stones beng processed. Snce these broken peces tend on average to be very small, the resultant partcle dstrbuton curve s typcally bmodal, rather than smply a mono-modal curve wth an extended small partcle zone. Some commercal supplers classfy the bare slca (that s, remove the fnes based on sze) pror to bondng, but agtaton n the bondng process can create more fnes. A lmted number of manufacturers offer SPE products that are fnes-free, and offer superor propertes compared to other supplers products. Partcle detals The frst problem that fnes create s they often fnd ther way nto the fnal sample extract. Ths occurs f the fnes are of a smaller sze than the porosty of the frt on the outlet of the SPE column. Ths frt s desgned to retan the sorbent wthn the column (and out of the extracted sample), yet wll not retan very small partcles. Crawford Scentfc 14

16 Fnes Channellng The second problem wth fnes has to do wth the bmodal sze dstrbuton n the sorbent. It s well known that bmodal sorbents do not dstrbute or mx evenly, but tend to aggregate; that s, the larger partcles group together among themselves, as do the smaller partcles. Ths results n effectve channels throughout the packed bed. These channels cause neffcences n vrtually every step of the extracton process, snce flow wll occur preferentally through the large partcle areas, due to the lower backpressure. The results nclude analyte breakthrough, and larger solvent volumes requred for every step n the extracton process. Crawford Scentfc 15

17 SPE Method Development Overvew The key to creaton of successful, rugged methods n SPE s well-planned, competentlyexecuted method development. There are many paradgms for method development n SPE n ths course an approach s presented that has stood the test of tme, s rapd, effcent, and results n very hgh-qualty sample extracts. Ths approach has the followng subsets: a. Analyte assessment durng ths step the analyte propertes are revewed and summarzed, ncludng functonal group chemstres that may be used for SPE, solublty, pka values of onzable groups, proten-bndng characterstcs, and prevously known chromatographc behavor. Analyte assesment Crawford Scentfc 16

18 b. Mechansm selecton n ths step a potental extractve mechansm s chosen, based on the results of the analyte evaluaton, and ncluson of further assay consderatons; for example, the nature of the raw sample matrx and the fnal analytcal procedure to be used. Mechansm selecton c. Sorbent screenng once a potental extractve mechansm s selected, sorbent columns exhbtng ths mechansm are screened for optmum analyte retenton and eluton, as well as degree of clean-up selectvty. d. Procedure optmzaton after a prelmnary protocol has been establshed, t s tested for ruggedness and optmzed to meet the overall requrements of the assay, ncludng cost, speed, potental for automaton, and other factors. Crawford Scentfc 17

19 Procedure Optmzaton Wash Solvent Strength e Method troubleshootng the fnalzed method s subjected to representatve usage, such as one or more complete sample batch extractons, after whch the method s revewed for vulnerabltes. Ths fnal evaluaton s recommended pror to method valdaton. Analyte Assessment I Method Troubleshootng The analyte assessment phase of method development s focused on predctng the behavor of an analyte throughout the extracton process. If the assay nvolves multple analytes, the process below should be carred out on each ndvdual analyte to be consdered, or the complete group of analytes together, f desred. Functonal group characterstcs drect the retentve behavor of the analyte relatve to sorbents. Analyte solublty nfluences wash and eluton solvent choces. Proten bndng of the analyte may affect necessary sample pre-treatment steps, as wll pka values of onzable groups. Fnally, prevously-known chromatographc behavor can ndcate retentve wndows for a Crawford Scentfc 18

20 partcular sorbent; that s, condtons of solvent strength under whch an analyte s retaned on or eluted from a gven chromatographc sorbent. The frst step of analyte assessment s a revew of the varous functonal group chemstres present on the analyte. It s recommended that ths process be carred out on paper, to provde a hard copy that may be referenced later as needed. Startng wth a structure of the molecule, the frst groups to be dentfed are non-polar or hydrophobc groups. These wll nclude any sgnfcant sectons of the molecule that are exclusvely hydrocarbon n nature. These may be hghlghted for reference, for example, by crclng them wth a red pen or pencl. Note that a sngle methyl group or ethyl group does not contrbute a sgnfcant amount of non-polar character typcally one s lookng for hydrocarbon moetes of 6 or more carbon atoms, ncludng saturated and aromatc rngs. Example of non-polar groups The next groups to be elucdated are polar groups. These nclude amnes, acds, amdes, ketones, aldehydes, esters, sulfhydryl groups, etc. Essentally any group contanng heteroatoms s a canddate polar group, except for ethers, whch are generally non-polar. It s recommended to hghlght these speces by crclng them wth a colour. Analyte Assessment II Example of polar groups The next phase of analyte assessment nvolves summarzng all other known propertes of the analyte, n addton to the functonal group exercse performed frst. Solublty characterstcs of a molecule can be of crtcal mportance to a successful extracton. Lqud/lqud extracton mplcatons asde, solublty s a factor n two prmary steps n an SPE procedure wash steps and eluton steps. Durng a wash step, t s desrable to use solvents that the analyte s not soluble n, f possble, snce these solvents wll typcally not elute the analyte from the sorbent surface, whle elutng nterferences. Durng eluton, a solvent must be employed wthn whch the analyte s soluble, or only ncomplete eluton may be the result. The best way to document solublty s smply to create a lst of solvents, and ndcate next to the lst whether the analyte s soluble or Crawford Scentfc 19

21 nsoluble n each respectve solvent. Even f the actual solublty data s not know, often the user can make an nformed guess based on the functonal group assessment performed prevously. As a smple example, an analyte that conssts almost exclusvely of amnes and hydroxyl groups wth vrtually no sgnfcant hydrocarbon content s unlkely to be hghly soluble n very non-polar organc solvents, but s very lkely to be soluble n water and very polar organcs such as methanol. Non-polar analytes are soluble n non-polar solvents Crawford Scentfc 20

22 Analyte Assessment III Polar analytes are soluble n polar solvents If proten bndng of an analyte s known or suspected, the user should be prepared to take the necessary steps descrbed earler n ths course to dsrupt the proten bndng. Often proten bndng characterstcs of an analyte are not known ntally and only become evdent durng the course of method development. One common ndcator of proten bndng often seen durng method development, s when a pure standard retans very well on an extracton sorbent, but the dentcal analyte from a spked matrx does not. If ths occurs, the user should then try the spked matrx extracton on a larger bed to confrm Crawford Scentfc 21

23 that the retenton problem s not a capacty ssue. If the larger bed mass equally fals to retan the analyte, proten bndng s very often the culprt. Crawford Scentfc 22

24 If analytcal work has been performed on the analyte prevously, partcularly HPLC, then somethng s lkely to be known about the analyte s chromatographc characterstcs, f only on a sngle packng materal. Even ths lmted data can be useful for example, f t s known that the analyte elutes n HPLC ar 80% organc n 20% buffer on a non-polar packng such as a conventonal C18, ths ndcates a strong retenton for the gven phase, and therefore relatvely aggressve wash solvents may be used n a non-polar extracton, to provde added cleanlness. Smlarly, ths same data would ndcate that a very strong eluton solvent s lkely to be requred. An addtonal analyte characterstc that should be noted s nstablty. If the analyte s known to be unstable under certan set of condtons; for example, a gven ph, then ths wll dctate lmtng condtons for the extracton. Ths s especally relevant n onexchange or mxed mode protocols, where ph changes may be essental to affect ether retenton or eluton. If the analyte s unstable under these condtons, a dfferent sorbent or mechansm choce may be necessary. Mechansm Selecton After performng the prevously-descrbed analyte assessment, the next step s to select an ntal extractve mechansm to work wth. The frst step n such a selecton s smply to revew the nformaton gathered, and summarze potental mechansm opportuntes. Typcally the analyte functonal group propertes provde the frst clues. For example, f an analyte has sgnfcant non-polar functonal groups but no onzable groups, a non-polar extracton mechansm s one obvous possblty, but on-exchange s not. On the other hand, f a molecule has many onzable and polar groups, but no sgnfcant non-polar character, then ether an on-exchange mechansm or a polar mechansm s a better choce than a non-polar mechansm. In cases where HPLC condtons are known for the analyte, a smlar mechansm s often chosen for extracton on the bass of avalable pre-exstng data or user comfort, yet ths approach wll tend to provde samples of poorer qualty than those obtaned from a mechansm orthogonal to the analytcal separaton mechansm, as descrbed earler. In general, t s recommended to temze all of the varous potental mechansms based on analyte propertes. Usually the revew of potental mechansms s lmted to sx potental categores: 1) nonpolar, 2) polar, 3) caton-exchange, 4) anon-exchange, 5) mxed mode, employng a nonpolar mechansm n combnaton wth ether a caton or anon-exchange mechansm, 6) chelaton or coordnaton chemstry. Retenton va mxed-mode requres that the analyte exhbt both non-polar and onc propertes for full realzaton of the mechansm potental. Crawford Scentfc 23

25 Crawford Scentfc 24

26 Mechansm Selecton After analyte propertes are consdered and potental mechansms recorded, the next step n mechansm selecton s to ncorporate other factors pertanng to the raw sample whch may elmnate lkely mechansms. There are many common stuatons whereby the matrx nature prevents facle use of a partcular mechansm for example, when workng wth a urne sample, even f the analyte s onzable, on-exchange may be an unrelable mechansm choce, because urne may contan hgh and varable salt content, whch can compromse analyte retenton through competton. Crawford Scentfc 25

27 Another example occurs when workng wth adpose tssue extracts n ths case there s a very hgh lpd content whch tends to saturate all avalable actve stes on a non-polar surface, thus nhbtng retenton of an analyte va a non-polar mechansm. Crawford Scentfc 26

28 As mentoned prevously, ph nstablty of an analyte may not prevent use of onexchange, but such nstablty may lmt the selecton of a partcular on-exchanger based on the ph the analyte would be subjected to n order to acheve eluton from the surface. Fnally, the overall nature of the sample matrx may dctate use of a partcular mechansm based on potental smplcty of the extracton procedure, as when aqueous samples are to be processed, use of a polar extracton requres that the analytes are frst transferred nto an approprate non-polar organc solvent from whch retenton can occur on a polar surface. In summary, the user should: 1. Start wth a lst of all avalable mechansms based on analyte structure and propertes. 2. Elmnate mechansms whch are unsutable for any partcular reasons. 3. Make a reasonable educated guess as to whch mechansm to start wth, understandng that other mechansms may be used n the event of falure wth the frst. Crawford Scentfc 27

29 Sorbent Screenng Once an extracton mechansm has been selected, a number of sorbents exhbtng the chosen mechansm should be screened for potental usage. Ths s often a stckng pont phlosophcally for new users, who feel that unnecessarly excessve work must be done n ths step. However, our experence has shown that a) well-organzed screenng s rapd and effcent, and b) any extra effort durng screenng s more than compensated by the flexblty allowed from dentfyng multple effectve sorbent choces. Many a user has gone far down the sngle sorbent path only to be blocked at the very end by a poor qualty extract, and forced to start over from scratch. For non-polar, polar and caton-exchange extractons, many dfferent sorbent choces exst, whle for anon-exchange, mxed mode, and chelaton the number of choces are sgnfcantly more lmted. Many commercal SPE supplers offer kts for sorbent screenng (look for Method Development Kts ), and these kts take much of the thought process out of screenng. They also allow use of many dfferent sorbent chemstres wthout nvestng n sngle boxes of each sorbent to be evaluated, whch can become expensve. Polar SPE method development kt Crawford Scentfc 28

30 Crawford Scentfc 29

31 Examples of approprate choces for each of the extracton mechansms are lsted below: a. Non-polar - C18 (multple varetes f avalable), C8, C4, C2, cyano and phenyl slcabased phases, and non-polar polymer phases. b. Polar - unbonded slca, amnopropyl, dol, non-endcapped short chan hydrocarbon (such as cyano, C2, C4 and phenyl) slca-based phases; and on-exchangers, both slca and polymer based. (Remember that on-exchangers typcally perform as polar phases n non-polar organc solvents.) Sorbent Screenng Alternatve Sorbents c. Caton exchange - weak and strong exchangers such as carboxylc acd, sulfonc acds n slca and polymer phases, plus non-endcapped short chan hydrocarbons n slca (such as cyano, C2, C4 and phenyl, n whch unbonded slanols act as caton exchangers). d. Anon-exchange - quaternary amnes, amnopropyl, dethylamno and damne phases, n both slca and polymer versons. e. Mxed mode - frst decde whch on-exchange mechansm to use (caton or anon) then screen all avalable mxed mode phases n both slca and polymer versons. f. Chelaton - these phases are typcally created by the user by startng wth ether an amnopropyl surface or a dcarboxylc acd sorbent, then treatng wth copper sulfate to gve a sorbent that wll complex amnes or carboxylc acd bearng analytes. Crawford Scentfc 30

32 Sorbent Screenng Alternatve Sorbents The actual steps employed n the screenng process are specfc to the respectve mechansm that s beng tested. In general, the screenng process s as follows: a. all of the sorbents are frst condtoned and pre-equlbrated dentcally. Crawford Scentfc 31

33 b. analyte standards n a matrx (modfed to make t sutable for the mechansm beng explored) are appled to the sorbents, and fractons collected to screen for breakthrough. Crawford Scentfc 32

34 c. sorbents exhbtng adequate retenton are then screened for eluton potental usng approprate solvents; fractons are collected to verfy proper eluton. Crawford Scentfc 33

35 d. analytes spked nto actual sample matrces are put through ntal protocols, and extracts analyzed to verfy adequate cleanlness. Crawford Scentfc 34

36 e. f clean-up s nsuffcent, wash steps are added to mprove extract qualty. Throughout ths process, careful notes should be taken, partcularly of solvents that do or do not elute analytes from the sorbents. Solvents that do not elute analytes may be very good canddates later n the process as wash solvents. Specfc detals as to solvents for condtonng, pre-equlbraton, washng and eluton, as well as sample propertes facltatng retenton, may be found n the earler sectons addressng the specfc mechansm n queston. Procedure Optmzaton The result of a well thought-out analyte assessment, mechansm selecton, and sorbent testng process s a prelmnary extracton procedure that gves adequate recovery and suffcent extract cleanlness to allow for target analyte analyss. At ths pont t s recommended to perform an addtonal process to optmze or tune the prelmnary procedure further. Ths optmzaton process helps to ensure that the fnal method s robust, rapd and cost effectve. A robust or rugged method s one whch can be readly used by multple extracton chemsts, allowng an approprate degree of varablty wthn sorbent surface chemstry and solvent protocols, yet stll provdng qualty extracts n a reproducble manner. Also, a rugged procedure must be usable on a suffcently wde range of samples of the type for whch the procedure s desgned. For example, a protocol for extractng sngle-speces Crawford Scentfc 35

37 plasma wll most lkely have less varablty requrements than a procedure for forensc human blood, snce the latter matrx wll vary n qualty to a much greater extent than the former. Therefore, durng method development, the forensc user must evaluate a much wder range of samples than the clncan. Procedure Optmzaton In order to test method ruggedness, t s necessary to start from a gven step of a procedure and test the roundedness of the parameters wthn the step; the man parameters beng solvent composton, solvent volume, and flow rate. So, for example, to test the ruggedness of an eluton step, the user should vary the composton of the eluton solvent to both weaker and greater strengths, vary the volume of eluton solvent used, and vary the flow rate of the eluton step. As a very specfc example, assume that a non-polar extracton has been developed that employs one mlllter of 80% acetontrle/20% water, appled at a flow rate of 1 mlllter per mnute, as the eluton step. To test the solvent composton ruggedness, a weaker solvent such as 70% acetontrle/30% water should be used, to verfy adequate eluton. Smlarly, a stronger solvent such as 90% acetontrle/10% water should be used, to verfy that the extract s stll suffcently clean. Eluton volumes both larger and smaller should be tested to dentfy the mnmum requred volume as well as the pont at whch larger volumes offer no addtonal beneft. For flow rate, t s recommended that at least % varance n flow rate should be tested, wth faster flow test beng the most mportant (snce slower flows very rarely have a negatve mpact on the qualty of a procedure). Crawford Scentfc 36

38 All of the above tests should be performed usng a representatve sample of a fxed sze. Once the optmzed procedure has been created, t s then recommended to test dfferent sample szes to verfy adequate performance. A second major factor affectng procedure ruggedness s the qualty of the manufactured SPE product. In partcular, the user should request multple sorbent lots for any protocol to be used over extended tme perods, snce a specfc lot of sorbent may only be avalable over a very short tme frame. Testng multple lots wll not ensure that any gven protocol wll transcend varablty n manufacturng chemstry, but successful testng of multple lots wll ncrease the chances for success dramatcally. Crawford Scentfc 37

39 Procedure Optmzaton The man factors contrbutng to the speed of an SPE procedure are the sze of the sorbent bed, the volume of solvent used n each protocol step (whch s somewhat nfluenced by the sorbent bed), the flow rate used to apply a protocol solvent, and the number of steps used n the procedure. To enhance procedure speed, t s desred to use a) the smallest sorbent bed possble whch gves adequate capacty, b) the smallest solvent volumes possble whch accomplsh ther msson, c) the fastest flow rates that do not cause deleterous effects, and d) the fewest steps possble. Therefore, the user should push the lmts of each of the above parameters to evaluate optmum condtons. Crawford Scentfc 38

40 Another major factor nfluencng procedure speed s the degree of sample pretreatment requred pror to the SPE protocol. For example, f a lqud/lqud extracton s requred to transfer analytes from an aqueous matrx to a solvent matrx n order to perform a polar extracton, ths s, n general, more tme-consumng than usng a mechansm whch can work drectly wth an aqueous matrx. On the other hand, there may be cases where a polar extracton provdes such superor extract qualty than a non-polar extracton, that the addtonal sample pretreatment may be well worth the tme. A procedure that has been optmzed for speed wll most often also be hghly costeffectve, snce small sorbent beds, reduced solvent volumes, fewer protocol steps, and less techncan tme expended all contrbute to cost benefts. Soak Steps Under certan crcumstances t may be necessary to nclude a soak step at varous ponts wthn the SPE protocol. Durng a soak step the solvent appled to the column s allow to reman statonary wthn the cartrdge and the flow rate and lnear velocty of the solvent fall to zero. Crawford Scentfc 39

41 The soak step allows tme for chemcal and physco-chemcal equlbra to be establshed between the solvent and sorbent bed and can greatly mprove retenton and eluton effcency and reduce the effects of varable flow rate through the cartrdge. Durng sorbent condtonng a soak step can be used to mprove the sorbent surface actvaton f the solvent s allowed to soak for (30 secs. to 2mns. for a typcal 100mg sorbent bed mass), pror to the equlbraton step. Ths approach s often used wth nonpolar sorbents that are hghly hydrophobc such as C18 (especally the end-capped phases) and for some C8 end-capped phases. Durng analyte eluton, the volume of eluton solvent, the number of solvent alquots requred and the rate at whch the eluton solvent s appled for successful analyte recovery can all be affected f a soak step s employed. Crawford Scentfc 40

42 By allowng a tme perod where the analyte speces can dffuse nto the extracton solvent and allow equlbrum to be reached, whlst the solvent s statonary, can be advantageous. The soak step wll ensure maxmum analyte recovery n the eluton solvent, may reduce the volume of extracton solvent requred and wll render the eluton step much less flow rate dependent. Crawford Scentfc 41

43 Dryng Steps Often a dryng step may be ncluded after the nterference removal / wash steps. Ths can be partcularly mportant when an aqueous sample has been loaded and the eluton step of the protocol nvolves manly water mmscble solvents. In ths case any water that remans on the surface of the sorbent may exclude the water mmscble eluton solvent and cause low recovery of the analyte. Polar sorbents generally requre longer dryng tmes than hydrophobc phases due to ther hgher wettablty. The dryng step may be sgnfcantly reduced or even elmnated when water mscble solvents are used for the eluton step. However, the presence of small amounts of water n the fnal eluate soluton can sgnfcantly ncrease the tme requred for solvent evaporaton (where analyte pre-concentraton s requred) or solvent elmnaton pror to resdue reconsttuton nto a solvent more sutable for the separatve stage of the analyss. Crawford Scentfc 42

44 Even small traces of water n the eluate soluton wll mean that to evaporate ths soluton to dryness wll requre extended heatng at hgher temperatures. Ths can be hghly unsatsfactory when the analytes are thermally or chemcally lable and analyte degradaton must be consdered. Eluton Optmsaton There are varous approaches to optmsng the applcaton of the eluton solvent wthn SPE protocols. The use of a soak step has already been detaled, however, the subject of eluton volume optmsaton s also worthy of consderaton. Once the eluton volume tself has been optmsed, ths step can be further optmsed by nvestgatng the applcaton of the eluton solvent n several small alquots. The followng experment hghlghts the varous advantages and dsadvantages of ths approach, the analyte s dazepam whch s beng eluted from a dsk format sorbent wthn a 96-well plate (C18-SD Hgh performance extracton dsk plate, 3M Company, St. Paul, MN, USA), the sorbent s C18 and varous eluton strateges are employed. The followng volumes are added to the cartrdge to nvestgate eluton volume requrements: 25, 50, 75, 100, 125 and 150 μl Crawford Scentfc 43

45 Eluton Optmsaton Results Each volume s added to eght (n=8) separate wells wthn the mcro-ttre plate contanng the dsk sorbent, to obtan an average recovery. Each tube s then subsequently eluted wth the same volume agan and the eluate collected nto a separate plate for analyss. The experment s then repeated a thrd tme, each cartrdge havng then been eluted wth three separate alquots to assess eluton effcency. As can be seen t would be possble to elute the dazepam wth as lttle as 25μL of eluent although workng wth such small volumes s dffcult and would almost certanly requre automaton. Ths volume would also requre three sequental elutons to derve maxmum recovery addng to overall processng tme for the plate. Eluton volume optmsaton wth sequental alquots 50 or 75μL eluton volumes appear more approprate, both yeldng almost complete recovery after only two alquots. Whlst the total recovery s smlar for both 50 and 75μL, the frst 75μL alquot s much more hghly concentrated (89% total recovery). In fact f senstvty s not an ssue, t would be possble to use just the frst alquot of 75μL as the standard devaton of the recovery for ths sngle alquot was good (s<2%), and not bother wth the second alquot. If protocol processng tme and mnmsng eluton volumes are an mportant facet of the method, t s recommended that the chemst nvestgates the eluton regme n a smlar Crawford Scentfc 44

46 fashon. Ths wll help to optmse recovery, mnmse eluton volume (perhaps negatng the need for further pre-concentraton) and maxmse sample throughput. SPE Method Troubleshootng Overvew Method troubleshootng s requred when, n the course of usng a prevously -valdated procedure, analyss falures occur. Proper due dlgence n the method optmzaton process prevously descrbed can help to mnmze the need for method troubleshootng, yet t stll happens that methods fal n the mdst of crtcal sample analyss. When ths occurs, a clear, concse approach to method troubleshootng and repar s essental. There are many categores of boanalytcal falure, ncludng poor reproducblty, nonlnear sample curves, loss of senstvty, chromatographc peak shftng, poor correlaton between nternal standard and target analyte extracton, etc. The full lst of potental problems and ther commensurate causes s beyond the scope of ths course. However, the very frst task n addressng method falures s to dentfy whch segment of the overall boanalytcal method s at fault; n other words, s the problem wth the analytcal separaton (for example, the chromatograph), the detector (perhaps a mass spectrometer), or wth the extracton method? Changng the extracton method to accommodate a detector problem s an extraordnarly neffcent use of the scentst s tme and efforts. Crawford Scentfc 45

47 Problems wth extracton methods tend to fall nto a lmted number of categores, ncludng a) poor recovery, b) poor reproducblty, c) poor cleanlness, and d) nadequate throughput. Each of these specfc problems has an assocated lst of troubleshootng technques and correctve actons. Problem solaton Recovery Problems I When low extracton recovery s perceved to be an ssue, the user should frst verfy that the problem s not caused by a change n the chromatographc system or the detector. The smplest dagnostc for ths s to nject known standards n fnal extract solvent to verfy that the response factor of the analytcal system has not changed snce the method was valdated. Then, absolute recoveres from the extracton procedure of both target analytes and nternal standards should be calculated, by comparson wth njectons of neat standards. Ths data may be compared to the orgnal absolute recoveres calculated when the method was frst valdated. Ths data wll help the user solate the problem to the extracton protocol or the analytcal system. Crawford Scentfc 46

48 Assumng the recovery problem s wth the extracton procedure, the next step s to verfy where the lost analytes have gone;.e., are they elutng from the extracton column and beng dscarded durng sample loadng or wash steps, are they remanng on the extracton column durng the eluton step, or are they lost n the sample pretreatment step? The best dagnostc to answer these questons s to process standards n pure solvents through the extracton procedure (as done n the orgnal method development), collect fractons from each protocol step, and look at each fracton analytcally, to determne whch fracton the analytes are n. Crawford Scentfc 47

49 Recovery Problems II If the analytes are breakng through the extracton column durng sample loadng or wash steps, frst verfy that the solvents used n these steps are correct, as well as the solvents used n condtonng and pre-equlbraton steps (snce these steps nfluence retenton). If these solvents all appear to be normal, and the breakthrough s a new behavor not seen prevously, ths suggests the SPE sorbent chemstry may have changed. Compare lot numbers of sorbent exhbtng the poor retenton wth the orgnal sorbents used, and contact the manufacturer to see f new lots may be obtaned. Crawford Scentfc 48

50 1. Verfy all solvents ntegrty usng alternatve technques use fresh solvents If changes to the method are allowed, try alterng the sample solvent or the wash steps to enhance retenton for the mechansm beng used (see detals n the respectve mechansm sectons). 2. Test dfferent sorbent lots (ncludng the orgnal on whch the method was developed) Crawford Scentfc 49

51 3. Investgate wash solvent strength 4. Investgate loadng solvent strength Crawford Scentfc 50

52 In the extreme case where the manufacturer s product has changed rrevocably, t may be necessary to change to a more retentve sorbent chemstry or even change the extracton mechansm used. 5. Change phase Recovery Problems III If the analytes are retanng on the column (as evdenced by ther absence from load and wash fractons) but not elutng, frst verfy that the eluton solvent s as t should be. If so, ths suggests that the sorbent chemstry may have changed. Agan, compare lots and contact the manufacturer to see f alternate lots exst or any data suggests changes to the product. If changes to the method are allowed, try alterng the eluton solvent to ncrease eluton strength for the mechansm beng used (see detals n the respectve mechansm sectons). Crawford Scentfc 51

53 Problems wth analyte eluton Also, revew the possblty of secondary nteractons between the sorbent and the analyte, and confrm that the eluton solvent addresses them. Investgate eluton solvent strength Crawford Scentfc 52

54 Fnally, t may be possble to change to a less retentve sorbent. Check for secondary nteracton and alter the eluton solvent accordngly Consder usng a less retentve phase Crawford Scentfc 53

55 If the SPE protocol appears to be workng properly, t s lkely that the analytes are never reachng the column durng the loadng step, or are breakng through durng loadng but are not detectable n the breakthrough fracton. The former may be caused by analyte nstablty n the sample, or f the sample pre-treatment nvolves proten precptaton and the analytes are proten-bound, they may be precptatng wth the proten fracton. Alternatvely, the proten-bound analytes may be passng through the SPE column unretaned, and are undetectable va the analytcal procedure n ther bound condton. In ether case, f proten bndng s suspected, try changng the sample ph, addng chaotropc agents, or dlutng the sample wth mechansm-compatble organc solvents, to dsrupt the proten bndng. Crawford Scentfc 54

56 Reproducblty Problems I As wth other method problems, the frst step n examnng rreproducblty s to dagnose the analytcal system to verfy that f s functonng correctly. Many tmes, reproducblty problems may be caused by sample-to-sample carryover, detector problems, or a defectve autosampler. Approprate experments to verfy correct nstrument operaton should be performed pror to alterng the extracton procedure for example, repeated njectons of pure standards, and/or njectons of standards of wde-rangng concentraton (especally very hgh levels followed by very low levels, to dagnose carryover). Crawford Scentfc 55

57 The most common causes of reproducblty problems n SPE are a non-rugged method, proten bndng problems, or changes to the sorbent from the manufacturer. Reproducblty Problems II If method optmzaton was performed orgnally as descrbe above, method ruggedness wll, deally, already have been confrmed. To examne current reproducblty ssues, frst confrm the ntegrty of all solvents used n the extracton protocol. If solvents are ntact and reproducblty problems stll occur, t s a smple matter to re-test the boundares of the protocol steps, by varyng the solvent composton and collectng fractons from analyte standards n approprate pure solvents. Any changes n method performance that may be exhbted for standards, as compared to the orgnal method performance, are most lkely a result of changes n the manufacturer s sorbent chemstry. Agan, the user s encouraged to contact the manufacturer and work wth them to resolve lot-to-lot problems. Crawford Scentfc 56

58 Test method boundares (ruggedness) aganst orgnal protocol If standards perform correctly, then t s lkely that the analytes may be experencng sample-related phenomena. The most common sample matrx-related phenomenon s proten bndng. Smlar steps to dsrupt ths should be appled as prevously descrbed. Cleanlness Problems Insuffcently clean samples may cause a number of analytcal dffcultes ncludng poor analyte quanttaton, poor reproducblty, or fundamental system problems such as hgh backpressure buld-up on the chromatographc column. One partcularly nsdous problem well-known to users of LC-MS s on-suppresson. Ths occurs when sample components co-elute wth the analytes nto the MS source, and nhbt onzaton of the analytes, reducng senstvty and often affectng lnearty of quanttaton. Ion-suppresson may be dffcult to dentfy, snce t does not show up as a chromatographc peak, but rather s evdenced ndrectly (as an analyte quanttaton problem). Crawford Scentfc 57

59 Ion suppressed sgnal Regardless of the symptoms of nsuffcently clean extracts, the approach to resoluton of the problem nvolves changng ether the wash protocols for the SPE sorbent, changng the sorbent to another usng the same extractve mechansm as the orgnal, or changng the extracton mechansm altogether. These three choces are lsted roughly n order of expermental prorty. The very frst tem to revew n the SPE protocol s the wash solvent. It s mportant to use a wash solvent that has as great an eluton strength as possble for the mechansm employed. Such a wash solvent wll elute the maxmum number of nterferences (but of course should not be so strong as to become an analyte eluton solvent). It s very common n SPE procedures that a user wll employ a wash solvent that s much weaker than necessary or approprate. Crawford Scentfc 58

60 Cleanlness Problems Alternatve Solvents Another approach to mprovng a wash step s to use a solvent that the analyte s nsoluble n. Ths approach s often overlooked snce t may nvolve use of solvents that are unfamlar to the user as SPE solvents. For example, n a non-polar extracton, most users wll work wth water-mscble solvents, typcally of weaker eluton strength than the analyte eluton solvent. However, dramatc mprovements n cleanlness may often be acheved by usng very non-polar, water-mmscble solvent (lke methylene chlorde, ethyl acetate, or hexane). Such solvents are, by defnton, very strong eluton solvents for the non-polar mechansm (.e., they wll elute many nterferences), yet analyte nsolublty wll keep the analyte retaned on the sorbent surface. The eluton solvent may also be nvestgated weakenng the eluton solvent strength may often result n cleaner extracts, however analyte recovery should be closely montored. Crawford Scentfc 59

61 Cleanlness Problems Poor Sorbent Selectvty If changng the wash solvents does not provde suffcent addtonal clean-up, changng the sorbent chemstry may help. The smplest approach, f possble, s to use a more selectve sorbent of the same mechansm as the orgnal. For example, f the method uses a C8 sorbent for a non-polar extracton, t may mprove clean-up to use a less-retentve sorbent, such as a C4 or C2. These sorbents tend to retan fewer matrx components than the C8, so more nterferences wll pass through durng sample applcaton. Of course, t s necessary that the analyte tself retan on the new sorbent. Crawford Scentfc 60

62 If changng the solvents and changng the sorbent wthn the current mechansm stll does not provde adequate clean-up, t may be necessary to change the extracton mechansm. Approprate alternates may be dentfed by revewng the nformaton ganed durng the method development process regardng potental usable mechansms (t s an unusual analyte ndeed that can be extracted usng only a sngle mechansm), One especally effectve technque s to go from a sngle extractve mechansm to a mxed-mode mechansm. Ths works best for analytes bearng both non-polar groups and onzable groups. Crawford Scentfc 61

63 Inadequate Throughput If a very large number of analytes are to be processed usng a gven method, t may occur that the speed of sample processng s not adequate. In ths case the user should revew all of the steps n the exstng protocol, and focus on: a) Reducng the sorbent bed sze Crawford Scentfc 62

64 b) Reducng volumes of solvent used n protocol steps c) Increasng the flow rate of solvent protocol steps Crawford Scentfc 63

65 d) Usng a wder sorbent bed of the same mass reduced lnear veloctes allow faster flow rates e) Explorng automaton. The user may also wsh to revew the sample pretreatment steps, snce these often represent a major tme element n the procedure Crawford Scentfc 64

66 If throughput s an especally crtcal element n a procedure, changng the extracton mechansm n the nterest of speed may even be justfed. A very hghly selectve SPE sorbent may reduce the number of solvent steps, solvent volumes, sorbent mass, etc., requred to obtan an approprate sample for analyss. All of the above-lsted technques have been addressed prevously n some detal. The sngle excepton s SPE automaton, whch wll be dscussed n an upcomng secton of the course. Crawford Scentfc 65

67 Generc Methods n SPE Overvew Method development s one of the more tme-consumng steps n the analytcal process. In addton, as seen through all of the materal presented so far n ths course, qualty method development requres a dscplned thought process and expermental program. However, when there s a shortage of avalable tme for proper method development and/or a shortage of approprate techncal knowledge to develop the necessary methods, generc methods may sometmes be used wth success. A generc method s a sngle protocol that delvers a reasonably clean extract for a wde varety of analytes a large percentage of the tme. Ths protocol commonly uses a sngle sorbent chemstry and a sngle solvent sequence whch the user may perform on many dfferent analyte samples. Such an SPE protocol by defnton must have three essental characterstcs: 1) the protocol wll allow for retenton of many dfferent analytes on the selected sorbent, 2) the wash steps n the protocol wll not elute the analytes from the sorbent, and 3) the eluton solvent wll successfully elute the analytes from the sorbent. Taken together, these three ponts provde clues to the man lmtaton of generc protocols that s, they often do not provde extracts of as hgh a qualty as custom-desgned protocols. Consderng each of the above three ponts n turn, 1) a sorbent that retans many dfferent analytes wll tend to retan many dfferent nterferences as well, 2) wash solvents that do not wash off the analytes tend to wash off fewer nterferences, and 3) eluton solvents that elute most analytes wll tend to elute a greater number of nterferences. Indeed, n the extreme, t mght be sad that a perfect generc protocol wll provde no sample clean-up whatsoever! Crawford Scentfc 66

68 In order to offset the cleanlness lmtatons of the perfect generc method, yet stll allow for rapd mplementaton of a successful protocol, we advocate a compromse approach. Ths approach begns wth a smple, generc protocol, but provdes rapd optmzaton for mproved extract qualty. Crawford Scentfc 67

69 Mxed Mode Sorbents Although the very frst attempts at generc methods nvolved non-polar sorbents, over tme these have been found, not surprsngly, to gve fnal extracts of relatvely poor qualty. Ths s due to the very non-selectve nature of the non-polar mechansm. However, a more advanced approach that has found consderable success and may be the most wdelyused generc technque today for pharmaceutcal applcatons, uses mxed-mode sorbents. As dscussed prevously, mxed mode sorbents are very powerful due to two prmary features: 1) multple retenton mechansms wll capture a wde range of molecular structure, and 2) the use of each ndvdual mechansm n sequence durng wash steps allows for very selectve clean-up. In other words, these sorbents offer the best of two worlds wde applcablty and hgh extract qualty. Crawford Scentfc 68

70 Most modern pharmaceutcal compounds contan at least one onzable functonal group (most often a basc group), and suffcent non-polar character that the compound may be retaned va a non-polar mechansm. Therefore, a typcal generc method for these compounds employs a mxed mode non-polar/on-exchanger. The specfc choce of onexchanger used depends on whether the analyte s basc or acdc. If the compound s basc, a mxed mode non-polar/caton-exchange sorbent s used, and f the compound s acdc, a mxed mode non-polar/anon-exchange sorbent s used. Generc Methods Overvew The startng pont for a generc mxed mode protocol s to frst select the sorbent. In general, the most common mxed mode sorbents are ether a polymer resn (nherently non-polar) wth caton or anon-exchange groups, or a slca base wth non-polar groups (most commonly C8, C18 or C4) and a caton or anon exchanger. These materals are readly avalable from the major commercal supplers of SPE sorbents. If the analyte s basc, select a sorbent contanng a caton exchanger; f the analyte s acdc, select a sorbent contanng an anon-exchanger. In addton, f the analyte s especally hydrophobc, a less retentve non-polar phase (such as a C4) may be used, and f the analyte s hghly polar, a more retentve phase (such as a C18) may be used. The procedure below may be used effectvely for serum, plasma, or urne. Crawford Scentfc 69

71 Generc Methods Basc Protocol The startng pont for the generc mxed mode protocol s as follows: 1.Condton the selected sorbent wth ether methanol or acetontrle. 2.Pre-equlbrate the sorbent wth a 50 mm buffer at a ph where the analyte and the sorbent are both charged. 3.Dlute the sample wth an equal volume of the equlbraton buffer n the prevous step. 4.Apply the sample to the sorbent at a flow rate of approxmately 1 ml per mnute. 5.Wash the sorbent wth the equlbraton buffer, but at 500 mm concentraton nstead of 50mM. 6.Wash the sorbent wth the orgnal 50mM equlbraton buffer. 7.Wash the sorbent wth the condtonng solvent (ether 100% methanol or 100% acetontrle). 8.Elute the analytes wth the condtonng solvent contanng 5% NH 4 OH (f the analyte s basc and a caton-exchanger s beng used), or the condtonng solvent contanng 5% HCl (f the analyte s acdc and an anon-exchanger s beng used). The ndvdual wash steps, as wth any mxed mode procedure, rely on the two separate mechansms to allow the analyte to reman on the sorbent whle nterferences that are ether solely onc or solely non-polar are beng eluted Crawford Scentfc 70

72 Generc Methods Further Optmzaton The above procedure s often adequate, provdng suffcent extract cleanlness and recovery. However, f further optmzaton s requred, ths should be done by 1) adjustng the wash solvents, and/or 2) adjustng the eluton solvent. The prncples here are the same as already descrbed many tmes, that s: 1) use the strongest wash solvent that does not elute the analytes, and 2) use the weakest eluton solvent that gves good analyte recovery. Specfcally, for wash solvents, try addng some organc (whch wll elute more nterferences that are non-polar n nature), or try usng dfferent ph buffers (whch wll elute more nterferences that are onc n nature). For eluton solvents, try usng less organc than 100%, so as not to coelute nterferences wth the analyte. Optmsed protocol Although these optmzaton steps wll by defnton add addtonal tme and reduce the beneft of the generc protocol concept, the total tme spent n method development usng ths overall approach wll stll typcally be less than f the more detaled approach descrbed earler s employed Crawford Scentfc 71