sample preparation for gc-ms...sample preparation for gc-ms 2 • objectives of sample preparation...
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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
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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
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3
Concentrate the analyte
Clean up the sample matrix
Analytical compatibility
Objectives of Sample Preparation
Not for use in diagnostic procedures
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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)
Not for use in diagnostic procedures
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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
Not for use in diagnostic procedures
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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)
Not for use in diagnostic procedures
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7
• Analytes, sample Matrix and SPE sorbent
• Chemistry of extraction protocols
• Troubleshooting and technical comments
SPE Fundamentals
Not for use in diagnostic procedures
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8
Extraction modes
• Nonpolar • Polar • Ion exchange • Covalent* • Affinity*
Format considerations
• Sample size • Processing speed • Device capacity
SPE Fundamentals
Not for use in diagnostic procedures
* Applies to liquid chromatography
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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?
Not for use in diagnostic procedures
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10
• Polarity • Metabolism • Concentration • Stability O
Analyte Fundamentals
Not for use in diagnostic procedures
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11
• What is the maximum concentration expected?
• What is the capacity of sorbent?
• Are there any matrix interferences?
Concentration
Not for use in diagnostic procedures
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
Run1Run2Run3Run4Run5
uL addedUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine
Shodex acetamide300171184038171653015793503456102601933060313332713399925996502573184101957450163100
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
Run1Run2Run3Run4Run5
uL addedUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine
Shodex acetamide3001947720048327021645000459040209269002285202090790053118020451400398740
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
uL addedUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine
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
uL addedUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamineUracilSalbutamolPseudo-ephedrinCaffeineMethamphetamine
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
Percent Breakthrough
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
Percent Breakthrough
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
Percent Breakthrough
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
Percent Breakthrough
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
Not for use in diagnostic procedures
-
13
• Silica based HyperSep cartridges
• Polymeric based SOLA cartridges
Log SP = c + mVx + rR2 + sπ2H + aΣα2H + bΣβ2H
Thermo Scientific™ HyperSep™ and SOLA™ SPE Sorbents
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
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
Not for use in diagnostic procedures
-
22
THC Sample After Derivatization
Urine sample normal GC/MS 16.78 min
Deuterated standard fast GC/MS 5.68 min
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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
Not for use in diagnostic procedures
-
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 petra.gerhards@thermofisher.com 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 analyze.eu@thermofisher.com and we will get back to you.
mailto:petra.gerhards@thermofisher.commailto:analyze.eu@thermofisher.com
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?
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