Sample Preparation for GC-MS...Sample Preparation for GC-MS 2 • Objectives of sample preparation • Types of sample preparation • Fundamentals for different sorbents • Polymeric

The world leader in serving science

Dipl.- Ing. Petra Gerhards Regional Marketing Manager Columns and Consumables, EMEA Thermo Fisher Scientific, Dreieich/Germany

Sample Preparation for GC-MS

2

• Objectives of sample preparation

• Types of sample preparation • Fundamentals for different sorbents • Polymeric Sorbents • Silica based Sorbents

• Fundamentals of solid phase extraction (SPE)

• Solid phase extraction products • Protocols • Real examples

Sample Preparation

Not for use in diagnostic procedures

3

Concentrate the analyte

Clean up the sample matrix

Analytical compatibility

Objectives of Sample Preparation


4

• Does not concentrate the analyte • Provides a cleaner extract than precipitation • Analytical compatibility is questionable

• Note every drug is extracted with the given/chosen solvent for LLE • The solvent is always for a large variety of drugs, special drugs like THC require

special sample preparation

Analytical compatibility

Liquid/Liquid Extraction (LLE)


5

LLE

Thermo Scientific™ HyperSep™ SPE cartridge

LLE extracts a lot of hormones and steroids, SPE is MUCH cleaner!

Liquid/Liquid Extraction (LLE) with Chlorobutane - Comparison


6

• Capable of concentrating the analytes • Up to 2ml of plasma or 3 ml of urine can be used,

compared to a 1:1 extraction with LLE • Concentration factor after SPE process is 1:60

• Yields very clean extracts

• Extracts are analytically compatible

Solide Phase Extraction (SPE)


7

• Analytes, sample Matrix and SPE sorbent

• Chemistry of extraction protocols

• Troubleshooting and technical comments

SPE Fundamentals


8

Extraction modes

• Nonpolar • Polar • Ion exchange • Covalent* • Affinity*

Format considerations

• Sample size • Processing speed • Device capacity

SPE Fundamentals


* Applies to liquid chromatography

9

H-Bond Donor

H-Bond Acceptor

Dipole

N

R1

R3

R2O

N R1

R2R2

R1

S

R1

O O

NR2R3

R3

O

HO R HO

R

O

HO S

O

O

R

O

H3C

R

X

N

C

RR

N+

O–

O R1

OR2

Hydrophobic

R

(E) (E)

R

R

aΣα2H

bΣβ2H

sπ2H

rR2

Log SP = c + mVx + rR2 + sπ2H + aΣα2H + bΣβ2H

How Does Chemistry in SPE Work?


10

• Polarity • Metabolism • Concentration • Stability O

Analyte Fundamentals


11

• What is the maximum concentration expected?

• What is the capacity of sorbent?

• Are there any matrix interferences?

Concentration


Method

Method

Preparation of solutions:

Prepare 5 drug working stock solution in pH 7.0 Phosphate buffer (0.025 M)

Low Conc StdMid Conc StdHigh Conc Std

mg in 500 mLug/mLng/uLmg in 500 mLug/mLng/uLmg in 500 mLug/mLng/uLLog P

Uracil1.252.502.506.412.8012.8058.7117.40117.40-1.07

Salbutamol1.252.502.5012.424.8024.80125.6251.20251.200.97

Pseudo-ephedrin1.252.492.4912.525.0025.00127.6255.20255.201.61

Caffeine1.252.502.5012.525.0025.00124.4248.80248.80-0.07

Methamphetamine2.505.005.002550.0050.00250.2500.40500.402.24

Tube Preparation

Pack 30 mg bed masses of each sorbent into VersaPlate tubes with

10 um bottom frits, 20 um top frits for 10 um experiments

or 20 um bottom frits, 20 um top frits for 20 um experiments

Sorbents used are:

Shodex Acetamide

Mitsubishi Acetamide

Tosoh Acetamide

Oasis HLB

Oasis MCX

SPE MethoduLapprox ugcapacity as% of 30mg bed mass

n=5 for each sorbentLow Conc3000.750.0025

Condition with 1 mL Methanol5001.250.0041666667

Followed by 1 mL water10002.50.0083333333

Apply 300 uL of Low Conc Std200050.0166666667

Collect eluate and inject on HPLCMid Conc3007.50.025

Apply 200 uL more of Low Conc Std (500 uL total)50012.50.0416666667

Collect eluate and inject on HPLC1000250.0833333333

Apply 500 uL more of Low Conc Std (1000 uL total)2000500.1666666667

Collect eluate and inject on HPLCHigh Conc300750.25

Apply 1000 uL more of Low Conc Std (2000 uL total)5001250.4166666667

Collect eluate and inject on HPLC10002500.8333333333

Repeat SPE method with new tubes20005001.6666666667

for both the mid(n=5) and high(n=5) conc stds

LC MethodTypical Retention Times (min)

Isocratic 10% ACN, 90% pH 7.0 Phosphate buffer (0.025M)Uracil0.7 - 0.8

5 cm 4.6 mm ID C8 SymetryShield (Waters)Salbutamol1.1 - 1.2

5 min run timePseudo-ephedrin1.5 - 1.7

1mL/minCaffeine2.2 - 2.3

Detection at 220 nmMethamphetamine2.9 - 3.0

50 uL injections for low and mid conc stds, 10 uL for high conc std.

capacity summary

UracilUracilUracilUracil

SalbutamolSalbutamolSalbutamolSalbutamol

Pseudo-ephedrinPseudo-ephedrinPseudo-ephedrinPseudo-ephedrin

CaffeineCaffeineCaffeineCaffeine

MethamphetamineMethamphetamineMethamphetamineMethamphetamine

Shodex acetamide

Toso acetamide

Oasis

MCX

ug

Capacity on Loading of 30 mg Cartridge

0.5

0.75

0.75

0.75

100

300

12.5

500

100

500

12.5

500

75

200

500

125

500

1000

100

1000

10 um data

Results for Low Concentration Sample

Run1Run2Run3Run4Run5

uL addedUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine

Shodex acetamide300426010216968340393230111530141803425820400925690452920163634131070459960120218360

500809190248900754020234300987020228100783570191900762960203604597

1000733510205301499828150162700588580177900850910147200841390198600

2000796610041580889880016050798990102014950852580033860800350032620

Mitsu acetamide300493525513016320407992378025250290010139314880332710153214067153359040232533242683

500710500168014120857480138200831720102200797200105100873890188400

1000578750000928600002710810800129600806850116800748770000

2000705490112600722800000837590119500702990000779160164500

Toso acetamide300285300011010199130010150208330114916170244370127610290185140164612040

500700050201000756250319928110764740294612093103751620136700851110289600

1000747010151900739120241700784020294811480830330284602067833300160216990

2000781570236602325813410194500529770011200725870223101021749880172300

Oasis300307490012391700270220010740297820100011770175240011620355200010090

500725480000818710001201654470000625060000637460000

1000712391206000825880000832352399000647270000845580000

200074696110850022027334415620006957921431129400699506036000766463957000

MCX300264060129701432214310137100215360000249650185801301194950000

500648790001529615450000486700000492330000605650000

100051730000057402000011150701447602260603120000643150000

2000681220000617210000645740000718000000669500000

Low Conc Std50 uL inj83709103927253141170001277761515846322335412430810612796609477818693108211134876909495577176871150447035393841168229914913428

Results for Mid Concentration Sample




50028287702026690602564990256257641122213316035557631025269301807364204804150000

100034428123420103801030573123300112271037503789032450330576603082803038057581035411712200801480

2000370889125321355857399702953751167903747522576034067782136248547718403581081509301942525088038087011940920405944760

Mitsu acetamide30011263501078001409930000213231371800015999402044001663850206200

50033648001050012473587850278600301592022800035274601951004116530103301488

1000358006004646038012501562866003919740069260382865001326703459180145128850

200036608131499034386903894623086402614540295639186270333699038196038294037909803819872605803114190

Toso acetamide3001036970000984180147400125503028030013839002810110501212240261100

50030857103623013473992400582600358685057220042829007655160520963817000527700

100035125700003634770263310030344841016211046033240702021003644500122700

20003746000024963035773001087171120397761001573103337500022103037466400147490

Oasis30017093577360001672750000172908652100015398801074001834180000

50030009715529355710032041854848000310377478790003682137368826840040481411233000

100032631250992637649097503630354325362302400310102465111211040033702651125633396044623799535681132291300

20003800819366648183905783639871992324185894056885366132937508995960543463840688587717654606783937640693888197738056768

MCX30010615000001456030001075139873000012109401086001061120000

50043369200004486260000434630000013806500004277400000

100025039500811702417400065880351000030518500001966850000

2000355413001509910333536001776150372708001783160351012001543280364190001173790

Mid Conc Std50 uL inj3306127792988505013382161538383839088459986944111243081339772742487462115938610302141004673298267543036467610536126445532934675580957473111714261656

Cannot integrate due to caffeine overload

Results for High Concentration Sample




500226525002085000229518001852180219212220302215760268666139301945980260587208602301250

10004401249983593524422298436108732068379222059740641662443813486046811486742894878230794515227915088578722282

20005304831650551330574100461506207166015128561211047954267110572711385748610848818317491713082201017135092041803891257542101096

Mitsu acetamide30017867600001611340000191499000017362600001879080000

500223557246501252180225705182701108000226006171001166200219523376801728730226653302501581490

1000564424103462082031328256576859112622082246126029612475158833093286629985554267111052083363430040545642102723082853529988

200050758916620613378721270174710271461661270889120220464194120053119075511284348808811790912211721143304872991276821191556112546

Toso acetamide30014263600001598520000156143000014830100001653700000

50033478800205803132380027580320809005835034295100003419290038320

1000328292001806910359996002806080336086002286230330663001866630336200002352360

20001629543544901397011018885222431013399870256384278930142356002008201824101340850016952128423012659380

Oasis30011223908376008410610861102300923221215118320089092582112191007955954871011500

500397771577674422201553040860761417474040262794371496869450361035668448943712335109702843648364110313160096025299

1000509252103464617130123131499525120711659800134689505137124846684390142630497829126675682040140719488400102838603050128759

200046849511836157405165293120850464042132691589000120577492344137222637083154541372564963481459226136528805112367847295617721171230246819135941

MCX30016132900001599630000182133000018028400001610090000

50032011200003148250000305871000031653500003370240000

1000321336006510090345474007016570323254007505940349211007756550345773006794690

200035277400131621303930110014747380474870001740609036073000136657903876860015044880

High Conc Std50 uL inj4894072158901095698123158489438727950119252512980648578110014601170526124534485868144127012856101356284735515510801203265139067

Cannot integrate due to caffeine overload

Averages

Shodex AcetamideMitsu AcetamideToso AcetamideOasisMCX


Low Conc30043158.802026.23533.2574.637665.41578.2105020751967.222445.40814.21193.2028119.402001132.234022766.60905.20546.6

50081935.202213.60919.481415.801403.8282.4076475.402483.6804620.669223.6000240.256978.4000305.8

100076850.801783.40299.877475.40492.8054278675.602266.4569.4413.477269.472100069053.202895.20452

200082768.202042781.2074960.60793.2007201001653224669.2728438814.2258.80440.466633.40000

Mid Conc300195627085621446.6799.8158647.6743.61036.800117446.401939.62210169704.82851.4214.800123766.40217.20215

500297160019904788.6224.4352251.2018200547375297.205620.6321688.6340783.868588.2165100376550.60000

1000337489.219592.4900.6131913.40371777.60602.67276.80351286.401500.4409.80343285.6497388.427617.202842.4198871.20029410

2000349183.8118917.22754.4538664.20363025.829068.40325907.803677010217.418931.60381081.291901388322.6058734.8355371.800155725.80

High Conc300206816.800420150178568.60000154460.4000091463.62721.810707.400168943.60000

500240901.61136.40208003.40224288.8255901367320330743002896.60435222.272448.450636026242.4318873.40000

1000440456.880275.20873463.417651.2570733.4117738.40847561.828859.6338247.400222364.20500028.6115706.864928.20133985.6337009.600711676.80

2000515349.6158420.601264104.499971.4483639.4135603.201242448.8117391.2195706.226487.401353469.20478837142281.462521.6203123.4127660.4393814.2001480525.40

Standardx axis (ng)

UracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineuL addedUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine

Low Conc72866945512037411274212576Low Conc300750.5714285714750747.78750.871499.904

Mid Conc329588776323.867205.21132698.4102878.65001250.952380952412501246.31251.452499.84

High Conc484809130000650001189524.8130438.610002501.904761904825002492.62502.94999.68

7875320005003.809523809550004985.25005.89999.36

Mid Conc300384074407500750015000

Integrations need work. Rough Estimate used for graphs500640012400125001250025000

10001280024800250002500050000

200025600496005000050000100000

High Conc30035220753607656074640150120

50058700125600127600124400250200

1000117400251200255200248800500400

2000234800502400510400497600.001000800

Shodex AcetamideMitsu AcetamideToso AcetamideOasisMCX


Low Conc30059.23%0.00%9.95%3.13%4.57%51.69%1.67%5.15%1.84%15.64%30.80%0.00%4.00%1.06%0.00%38.59%0.00%0.98%1.00%2.70%31.24%0.00%4.44%0.00%4.35%

500112.45%0.00%10.86%0.00%7.31%111.73%0.00%6.89%0.25%0.00%104.95%0.00%12.19%0.71%4.93%95.00%0.00%0.00%0.00%1.91%78.20%0.00%0.00%0.00%2.43%

1000105.47%0.00%8.75%0.00%2.38%106.33%0.00%2.42%0.00%4.31%107.97%0.00%11.12%0.51%3.29%106.04%0.76%0.00%0.00%0.00%94.77%0.00%14.21%0.00%3.59%

2000113.59%0.00%1.00%2.47%0.00%102.87%0.00%3.89%0.00%0.00%98.82%0.00%8.11%0.20%5.32%99.97%9.32%1.27%0.00%3.50%91.45%0.00%0.00%0.00%0.00%

Mid Conc30059.36%0.00%12.74%0.13%0.78%48.14%0.10%1.54%0.00%0.00%35.63%0.00%2.89%0.02%0.00%51.49%0.37%0.32%0.00%0.00%37.55%0.00%0.32%0.00%0.21%

50090.16%0.00%2.96%0.42%0.22%106.88%0.00%2.71%0.00%0.53%113.87%0.00%8.36%0.03%0.67%103.40%8.83%2.46%0.00%0.00%114.25%0.00%0.00%0.00%0.00%

1000102.40%2.52%1.34%11.65%0.00%112.80%0.00%0.90%0.64%0.00%106.58%0.00%2.23%0.04%0.00%104.16%64.07%41.09%0.00%2.76%60.34%0.00%0.00%0.26%0.00%

2000105.95%15.32%4.10%47.56%0.00%110.15%3.74%0.00%28.77%0.00%111.56%0.00%0.32%1.67%0.00%115.62%118.38%131.42%0.00%57.09%107.82%0.00%0.00%13.75%0.00%

High Conc30042.66%0.00%0.00%3.53%0.00%36.83%0.00%0.00%0.00%0.00%31.86%0.00%0.00%0.00%0.00%18.87%2.09%16.47%0.00%0.00%34.85%0.00%0.00%0.00%0.00%

50049.69%0.87%0.00%17.49%0.00%46.26%1.97%0.00%11.49%0.00%68.22%0.00%0.00%0.24%0.00%89.77%55.73%77.90%0.00%20.12%65.77%0.00%0.00%0.00%0.00%

100090.85%61.75%0.00%73.43%13.53%117.72%90.57%0.00%71.25%22.13%69.77%0.00%0.00%18.69%0.00%103.14%89.01%99.89%0.00%102.72%69.51%0.00%0.00%59.83%0.00%

2000106.30%121.86%0.00%106.27%76.64%99.76%104.31%0.00%104.45%90.00%40.37%20.37%0.00%113.78%0.00%98.77%109.45%96.19%17.08%97.87%81.23%0.00%0.00%124.46%0.00%

Capacity in ug

Log P-1.070.971.61-0.072.24

UracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine

Shodex acetamide0.510010075500

Mitsu acetamide0.512512575300

Toso acetamide0.753005002001000

Oasis0.7512.512.5500100

MCX0.755005001251000

Shodex 10 um

750.5714285714750.87

1250.95238095241251.45

2501.90476190482502.9

5003.80952380955005.8

64007500

1280012500

2560025000

11740074640

234800124400

248800

497600

Uracil

Caffeine

ng

Percent Breakthrough

0.592302055

0.0313386978

1.1244609984

0

1.0546837903

0

1.1358929106

0.0246686251

0.9016104955

0.0012771273

1.0239729602

0.0042276037

1.0594554413

0.1164594211

0.9085161373

0.0353208273

1.0629951177

0.1748626006

0.7342960819

1.0626969694

Mitsu 10 um

750.5714285714750747.78750.871499.904

1250.952380952412501246.31251.452499.84

2501.904761904825002492.62502.94999.68

5003.809523809550004985.25005.89999.36

640074407500750015000

1280012400125001250025000

2560024800250002500050000

117400496005000074640100000

2348007536076560124400150120

125600127600248800250200

251200497600500400

5024001000800

Uracil

Salbutamol

Pseudo-ephedrin

Caffeine

Methamphetamine

ng


30 mg Mitsubishi AcetamideCapacity Study

0.5169118192

0.016691521

0.0515362717

0.0184047883

0.1564249364

1.1173328649

0

0.0689015412

0.0025048252

0

1.0632556659

0

0.0241876902

0

0.0430979644

1.0287430935

0

0.0389319721

0

0

1.0687622122

0.0009578477

0.0154273776

0

0

1.1280070876

0

0.0270812378

0

0.0053169464

1.1014533296

0

0.0089665681

0.006424305

0

1.1772335085

0.0374436543

0

0

0

0.9975875035

0

0

0.1149467418

0

0.0196846154

0

0.7125213363

0

0.90568

1.0444917164

0.2212504581

1.0431015385

0.8999728608

Toso 10 um

750.5714285714750747.78750.871499.904

1250.952380952412501246.31251.452499.84

2501.904761904825002492.62502.94999.68

5003.809523809550004985.25005.89999.36

640074407500750015000

1280012400125001250025000

2560024800250002500050000

496005000050000100000

753607656074640150120

125600127600124400250200

251200255200248800500400

5024005104004976001000800

Uracil

Salbutamol

Pseudo-ephedrin

Caffeine

Methamphetamine

ng


30 mg Tosoh AcetamideCapacity Study

0.3080358246

0

0.0399626976

0.0105834185

0

1.0495318817

0

0.1219004614

0.0071313011

0.0493479644

1.0797269516

0

0.1112398155

0.0050504513

0.0328721374

0.988249696

0

0.0811328163

0.0019868302

0.0532124682

1.1386858745

0

0.028860862

0.0001951093

0

1.0658349212

0

0.0836334093

0.0002833941

0.0066933259

1.1156383121

0

0.0223256534

0.0003617909

0

0

0.0032348687

0.0167137166

0

0

0

0

0

0

0

0.00243509

0

0

0

0.1869353207

0

0.2037492308

0

1.1378234401

0

Oasis 10 um

750.5714285714750747.78750.871499.904

1250.952380952412501246.31251.452499.84

2501.904761904825002492.62502.94999.68

5003.809523809550004985.25005.89999.36

640074407500750015000

1280012400125001250025000

2560024800250002500050000

11740012560012760050000150120

23480025120025520074640250200

502400510400124400500400

2488001000800

497600

Uracil

Salbutamol

Pseudo-ephedrin

Caffeine

Methamphetamine

ng


30 mg OasisCapacity Study

0.3859045758

0

0.0098164327

0.0100423621

0.0270356234

0.9500097439

0

0

0

0.0190998728

1.0604285663

0.0076255143

0

0

0

0.9996816082

0.0932216476

0.0127024639

0

0.035019084

1.0339690765

0.0036729519

0.0031961812

0

0

1.0415597655

0.08834999

0.0245665514

0

0

1.1562350571

0.6406970906

0.4109384393

0

0.0276286808

1.0313929816

0.5572953846

0.7790153846

0

0

0.9876817468

0.8900523077

0.9988953846

0

0.2011858453

1.0944723077

0.9618707692

0

1.0271928708

0

0.97870109

0.170760122

MCX 10 um

750.5714285714750747.78750.871499.904

1250.952380952412501246.31251.452499.84

2501.904761904825002492.62502.94999.68

5003.809523809550004985.25005.89999.36

640074407500750015000

2560012400125001250025000

24800250002500050000

496005000050000100000

753607656074640150120

125600127600124400250200

251200255200248800500400

5024005104004976001000800

Uracil

Salbutamol

Pseudo-ephedrin

Caffeine

Methamphetamine

ng


30 mg MCXCapacity Study

0.312443904

0

0.0444291744

0

0.0434637405

0.7819592623

0

0

0

0.0243161578

0.9476712111

0

0.1421026799

0

0.0359414758

0.9144623982

0

0

0

0

1.1424888042

0

0.0032318928

0

0.0020898418

1.0782303967

0

0

0

0

0

0

0.0025964546

0

0

0

0.13748214

0

0

0

0

0

0

0

0

0

0

0

0.5982866435

0

0

0

1.2446360093

0

HPLC ACN

-1.07-1.07-1.07-1.07-1.07

-0.07-0.07-0.07-0.07-0.07

0.160.160.160.160.16

1.610.970.970.970.97

1.831.611.611.611.61

2.21.831.831.831.83

2.242.22.22.22.2

3.442.242.242.242.24

4.23.443.443.443.44

4.64.24.24.24.2

4.644.64.64.64.6

5.184.644.644.644.64

5.185.185.185.18

Uracil

Caffeine

Salbutamol

Pseudo-ephedrin

Methamphetamine

4 - 5% acetamide

Oasis HLB

Oasis MCX

4 - 5% acetamide

4 - 5% acetamide

Log P

Log K'

Retention in 60% ACN pH 7.0 Phosphate

-0.1168208464

-0.7629112838

0.0117384033

-0.0263289387

-0.2775488998

-0.04758226

-0.3301882244

-0.2722582531

-0.1055101848

-0.2138798199

0.6365068203

-0.7508281232

2.036278799

1.2109843545

0.9481683617

0.9061520054

-0.4778268511

2.1791738973

1.3565132599

1.1663314218

1.0578382779

-0.3853173101

2.4398299818

1.434506285

1.2420027813

0.244207079

-0.1023491111

2.1435708478

1.5859028721

1.3889198159

1.0417649908

0.3509273309

0

0.2490784031

0.0821867562

1.2981525016

-0.3266838119

2.5697906374

1.5778746068

1.3807136067

1.2081978951

0.2450477445

2.1430575984

1.9108963161

1.6215224711

0.7381221283

0.8969661869

2.0648168468

1.8997239675

1.4086638741

1.3278406809

0.5526445172

1.2698965966

1.1979558723

0.9615529182

1.4656353401

0.70730283

2.3358012545

1.8746068616

1.6445479973

1.2052348377

2.6888950135

2.0775816329

1.9537060122

HPLC MeOH

-1.07-1.07-1.07-1.07

-0.07-0.07-0.07-0.07

0.160.160.160.16

0.970.970.970.97

1.611.611.611.61

1.691.691.691.69

1.831.831.831.83

2.112.112.112.11

2.22.22.22.2

2.242.242.242.24

2.522.522.522.52

2.942.942.942.94

3.443.443.443.44

4.24.24.24.2

4.64.64.64.6

4.644.644.644.64

5.185.185.185.18

Uracil

Caffeine

Salbutamol

Pseudo-ephedrin

Methamphetamine

4 - 5% acetamide

4 - 5% acetamide

Oasis

Oasis MCX

Log P

Log K'

Retention in 60% MeOH pH 7.0 Phosphate

-0.1953964143

0.3094089272

-0.2918866162

-0.0334237555

-0.0754210972

0.4167053322

0.0562559241

-0.4536401589

1.1514897641

1.4843922916

-0.7690078709

1.9753466945

1.2009614634

1.4622034651

-0.3729538244

1.9538849182

1.4496237099

1.7139376006

0.0565782438

2.4337402105

0.71745341

0.6006476503

1.1110908331

0.967443966

1.8361038573

2.1706045644

1.0684377774

2.4057825798

0.7854330051

0.7429188739

1.3438902474

1.0662568856

0.1296338578

0.5301497822

0.7595658661

0.6405182431

1.7772061042

2.0350917152

0.4518669637

2.6716355966

0.9185469381

0.8843612865

1.6618529465

1.2783301056

1.1594102477

1.0706374725

2.0131959235

1.507155961

1.7471752916

2.2209227723

1.5032689731

2.293198809

1.6371124985

2.5550001734

2.9692820461

2.597532681

1.5462206856

2.1680457275

2.781495652

2.1941030295

2.3958200175

2.8101451998

2.627540167

3.0903691048

2.750284745

3.0508516082

2.9294515373

3.6477197546

12

• Capacity is dependent on sorbent type, bed volume and chemistry

• General rule is 4 – 10% of the bed volume on non-polar chemistries

• Certificates of analysis generally indicate sorbent capacity

Sorbent Capacity


13

• Silica based HyperSep cartridges

• Polymeric based SOLA cartridges

Log SP = c + mVx + rR2 + sπ2H + aΣα2H + bΣβ2H

Thermo Scientific™ HyperSep™ and SOLA™ SPE Sorbents


14

• Non-polar (Hydrophobic) phases • C18(ec), C18, C8, C4 etc.

• Polar and ion-exchangers

• CN, Phenyl, SAX, NH2, SCX, SI

• Mixed phases • C8 / SCX

Silica Based Sorbents


15

• Sample pre-treatment Right pH? • Conditioning Is the flow slow enough to activate the sorbent? • Sample application pH correct, flow correct? • Washing Break through? • Elution Solvents correct, enough elution steps?

Troubleshooting


16

If the substance contains NH or NH2 or OH groups

• Recommend HyperSep Verify, SCX or WCX will work for these functional groups

• If the substance is a little bit more polar (containing OH groups), recommend SCX

• If the substance is a little bit more non polar, recommend WCX

For ion exchange mechanisms the target is to charge the functional groups.

HyperSep Based On Cation Exchanger Mechanism


17

Morphine SCX or Verify

Amphetamine WCX or Verify

Diazepam WCX or Verify

For a broad extraction of Drugs of Abuse Verify For a specific extraction of groups SCX or WCX

Basic Drugs, Where Charges Can Be Applied


18

If a compound contains NH or NH2 plus CO, OH or COOH groups recommend HyperSep WCX if the NH or NH2 group is not steric hindered.

If it is steric hindered, use HyperSep C18 or HyperSep C18 acid wash, C8 can also be an option. A method development set would be a good choice, containing HyperSep C18, C8 and Verify.

Steric hindered N in Ecgonine Alakloide from Cocain and Benzoylecgonine, main metabolite

Zwitterions


19

THC carboxylic acid More hydrophobic interaction

Mostly hydrophobic C18 For GC sample has to be evaporated and derivetized and reconstitution is done on Hexan: Ethylacetate 75:25

Ibuprofen, acidic drug C18 or Verify AX

Acidic Drugs


20

400 µl Methanol let it react for 2 min. Equili. with 400 µl 0.1 M acetic acid

200 µl plasma dilt. 1:1 with dist. water, add 20 µl acetic acid, pH 4

400 µl 0.1 M acetic acid 200 µl methanol Dry with strong vacuum 2 min.

Elution Condition

Interference- elution

2 x 200 µl NH3/methanol 1 : 9

Cation Exchanger SOLA 10mg/1ml For Plasma Generic Protocol


Low volumes only!

21

400 µl Methanol let it react 2 min. Followed by 400 µl neutral buffer Ammonia acetate, in some cases water will be enough

200 µl plasma dilt. 1:1 with buffer or dist. Water ,

400 µl buffer + 3 % isopropanol 400 µl buffer Dry with strong vacuum 2 min.

Elution Condition

Interference- elution

2 x 200 µl

Buffer * + ACN Evaporate, derivetize and reconstitute in Hexan 75:Ethylacetate 25

SOLA HRP 10mg/1ml For Plasma THX Example


22

THC Sample After Derivatization

Urine sample normal GC/MS 16.78 min

Deuterated standard fast GC/MS 5.68 min


23

3 Ml urine +30 µl

ß-Glucuronidase

Incubate at 56 °C for 60 min

Conditioning with 3ml methanol

pre-equilibration with

diluted phosphoric acid

Sample application

Interference elution with 3 ml

Diluted phosphoric acid, 0.5 ml 0.1 N acidic acid and

1ml methanol

Analyte elution with

2 x 2 ml

NH3/methanol

HyperSep Verify 200 mg For Urine Generic Protocol


24

3.8. 11.20 Uhr Crea 45.2 mM/l 3.8. 18.45 Uhr Crea 3.6 mM/l 4.8. 10.00 Uhr Crea 14.7 mM/l

Without SPE you can not determine SPICE ME, concentration is too small, within 24 hrs the ME are already excreeded

SPICE Urine WITH ME OF Synthetic Cannabinoides


25

Any Questions?

Do you want to receive detailed SPE protocols? We have protocols ready for drugs of abus analysis. Others could be assembled upon request. Please send an e-mail to [email protected] and specify if required for GC or LC. Do you have additional questions or do you want to talk to an expert from Thermo Fisher Scientific? Please send an E-Mail to [email protected] and we will get back to you.

mailto:[email protected]:[email protected]

Sample Preparation for GC-MSSample PreparationObjectives of Sample PreparationLiquid/Liquid Extraction (LLE)Liquid/Liquid Extraction (LLE) with Chlorobutane - ComparisonSolide Phase Extraction (SPE)SPE FundamentalsSPE FundamentalsHow Does Chemistry in SPE Work?Analyte FundamentalsConcentrationSorbent CapacityThermo Scientific™ HyperSep™ and SOLA™ SPE SorbentsSilica Based SorbentsTroubleshootingHyperSep Based On Cation Exchanger MechanismBasic Drugs, Where Charges Can Be AppliedZwitterionsAcidic DrugsCation Exchanger SOLA 10mg/1ml For Plasma Generic ProtocolSOLA HRP 10mg/1ml For Plasma THX Example THC Sample After DerivatizationHyperSep Verify 200 mg For Urine Generic ProtocolSPICE Urine WITH ME OF Synthetic CannabinoidesAny Questions?

Documents

Sample Preparation for GC-MS...Sample Preparation for GC-MS 2 • Objectives of sample preparation • Types of sample preparation • Fundamentals for different sorbents • Polymeric