Cryogenic Zone Compression for the Measurement of Dioxins in Human

S t Att L l b GC GCSerum at Attogram Level by GCxGC-IDHRMSIDHRMS

D.G. Patterson Jr.1, S.M. Welch1, W.E. Turner1,D.G. Patterson Jr. , S.M. Welch , W.E. Turner , J.-F. Focant2

1CDC, NCEH, DLS, OATB

2MS LaboratoryBiological and Organic Anal. Chem.

Estimated SituationFrequencyFrequency

A considerable proportionof the general population A considerable proportionof the general population g p p

exceeds the TDIg p p

exceeds the TDI

20 20 Intake (pg TEQ/kg BW/day)

TDI

20 Intake (pg TEQ/kg BW/day)

TDI

20

Source : EFSA 2006TDITDI

Based on 29 molecules

Various Strategies Emission (environmental) control, use of

complex mathematical modelcomplex mathematical model

f f (f d) f l i

evel

s

Measurement of food (feed) for regulation

asin

g l

Sample humans directly : BIOMONITORING Dec

rea

BIOMONITORING• Adipose tissues

B ilk

D

• Breast milk• Serum

Complex Matrix

Temporal decrease1980’s

MatrixHeavy sample preparation

3

‘Cleaned’ Milk Extract3

2

2tR (s)

1

29002400190014009000

1tR (s)

1-20ml available Sub-ppt level

5ml, 0.5% fat, 1ppt = 25 fg/5g serum5ml, 0.5% fat, 1ppt 25 fg/5g serum~ 15 fg injected (60% recovery)

SensitivitySensitivitySensitivitySensitivitySensitivitySensitivity

SpeedSelectivity SpeedSelectivity SpeedSelectivity(Low cost)

Selectivity(Low cost)

Selectivity(Low cost)

Selectivity

QA/QC Guidelines EU Directive 2006/13/EC & 2006/88/EC EU Regulation (EC) 1883/2006 & 152/2009 EU Regulation (EC) 1883/2006 & 152/2009 EURACHEM Guide ‘The fitness for

purpose of analytical methods’

Requirements for analytical procedures:-Sensitivity (LOQs) -Selectivity-Accuracy -Trueness-Robustness -13C-labeled IS-Recovery rates -Good separation

A Method is Mainly Defined by :

The target measurement level (the regulation) The target measurement level (the regulation)

The sample amount available

The sample-prep reasonable load

Sample dimensionality (complexity)

Th l il bl The tools available

Other issues (blanks throughput versatility ) Other issues (blanks, throughput, versatility…)

The Analytical Warehouse (HR)GC-IDHRMS The gold standard (HR)GC-IDQISTMS/MS Outsider (HR)GC-IDQISTMS/MS Outsider FGC-IDTOFMS Outsider ( )GC GC O S O id (HR)GCxGC-IDTOFMS Outsider RBA, EIA Outsider others… Outsider

(HR)GC-IDHRMS is still the mosti i l il bl dsensitive tool available today…

(HR)GC-IDHRMS Selected ion monitoring (SIM)

only specified ions are recorded inonly specified ions are recorded in defined retention windows (sensitivity enhancement increased dwell timeenhancement, increased dwell time, segmentation of the GC run)

Quantification by internal std isotope dilution Quantification by internal std , isotope dilution13C-labeled standards for each separate congener (best practice in anal Chem )congener (best practice in anal. Chem.)

NHANES 2005 reportNHANES 2005 report

Aim of the Work Take the best MS tool for dioxins = HRMS

Take the best method for dioxins = 13C ID

Take the best GC signal enhancer = GCxGCTake the best GC signal enhancer GCxGC

Make the coupling

Face the challenges

See what happenSee what happen…

(PS : This is a 15 year old idea…)

1,547398,45

1,0471401,45

0,5471404,45

0,0471407,45

2,5471407,45

2,0471410,45

1,5471413,45

1,0471416,45

0,5471419,45

Organohalogen Compounds, 1996, 27, 309

Experimental GCxGCAgilent 6890N GC

Loop Modulator (Zoex Corp.), Liquid N2

DB-5MS (0.1mm ID x 0.1μm df)DB 5MS (0.1mm ID x 0.1μm df)

1D : 6m, 2D 75cm, PM 5-9s, hot pulse 0.6-08s

MAT95XP & DFS (Thermo) HRMS

XCalibur (Thermo) + GC Image (Zoex) XCalibur (Thermo) + GC Image (Zoex)

EI Mode at 10,000 RP

Standard solutions and real serum extracts

Loop ModulatorDelay Loop

Hot JetCold JetCold Jet

Zoex Corp.

HRMS Tuning 300-400 ms peak width at the base (no 2D oven) 1 SIM group per congener class1 SIM group per congener class Cycle times from 40 to 70 ms (14 to 25 Hz)

TCDD (+ others) window (HR)GC-IDHRMS GCxGC-IDHRMS

Number of ions 15 3Number of ions 15 3Target dwell time (ms) 15-100 10Lock mass dwell time (ms) 50 2Electric jump time (ms) 10 6Electric jump time (ms) 10 6Cycle time (ms) 1100 44Scan rate ~0,5Hz ~20Hz

Approximately 7 data points per peak

12C-2378-TCDD Standard

9 13

80

90

1009.13

1 fg on-column

S/N 142030 ms cycle time (33Hz)

60

70S/N 142030 ms cycle time (33Hz)

m/z 321.8936 [M+2] only

30

40

50

0

10

20

8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 .39 9.4 9.5 9.6 9.7 9.8

Time (min)

12C-2378-TCDD Standard313 ag 12C-2378-

TCDD

(S/N>400, 4 Sigma)

Maximal sensitivityy

Linear calibration :0 313 0 625 1 250.313, 0.625, 1.25, 2.5, 10, 20 fg/μl

m/z 321.8936 [M+2] only

Real Serum Samplesp NIST SRM 1589a human serum

1-5-10g aliquots prepared with routine method

R i d i 5 20 l f Reconstituted in 5-20 μl of nonane

Further diluted to reach concentrationsFurther diluted to reach concentrations

(assuming 70% recovery rates) :

6.5, 1.6, 1.3, 1.1, 0.81, 0.65, 0.54, 0.41, 0.32,

0 20 0 16 f / l0.20, 0.16 fg/μl

12C-2378-TCDD Serum540 ag 2378-TCDD, assuming 70 % recovery

S/N 474 (4 Sigma)( g )

Diluted 20g sample

m/z 321.8936 [M+2]m/z 319.8965 [M]

Significant deviation from the theoretical isotope ratio…

12C-12378-PeCDD Serum223 ag 12378-PeCDD, assuming 70 % recovery, S/N 188 (4 Sigma)

m/z 355.8546 [M+2]

Diluted 20g sample

12C-2378-TCDD SerumConventional GC-IDHRMS

Approx. 4 fg on columnGCxGC-IDHRMS

Approx. 4 fg on column90

100100

50

60

70

80

50

60

70

80

90

Peak height 1010

m/z 321.8936 m/z 321.8936

10

20

30

40

10

20

30

40

50 1010

S/N 601Diluted 20g sample

70

80

90

100

70

80

90

100

m/z 333.9339 m/z 333.9339

30

40

50

60

30

40

50

60

70

Peak height 433,319

Peak height 42,373

6.4 6.6 6.8 7.0 7.2 7.6 7.8

10

20

3.58 3.60 3.62 3.64 3.66 3.68 3.70 3.72 3.74Time (min)

10

20

Time (min)7.4

PCDDs/PCDFs/cPCBsOcta Dioxin

Hexa Dioxins

Hepta Furans and Dioxin

Penta Dioxin

Hexa Dioxins (unresolved)

Tetra PCB

Tetra Dioxin

Total Ion ChromatogramTotal Ion Chromatogram

1 µL injection of 4g NIST SRM1589a Serum Sample ExtractSample Extract

Non-diluted 4g sample

m/z 321 8936 [M+2]m/z 321.8936 [M+2]

Noise + ion ratio skewingNoise + ion ratio skewing…

Possible StrategyLow level sample

Sample preparation

GCxGC-IDHRMS GC-IDHRMS(Selected Descriptors) (Multi-Group)

2,3,7,8-TCDD… 17 PCDD/Fs + NO-PCBs

Report with undesirable ‘not detected’Report with NO ‘not detected’

Challenges (I) Ion ration skewing strange masse responses

Quantity Notation Number of moles Number of molecules6 9 15

Ion ration skewing, strange masse responses, …

1 microgram (µg) or 10-6 g ppm 3 nanomoles or 3.10-9 2,000,000,000,000,000 (2.1015)1 nanogram (ng) or 10-9 g ppb 3 picomoles or 3.10-12 2,000,000,000,000 (2.1012)1 picogram (pg) or 10-12 g ppt 3 femtomoles or 3.10-15 2,000,000,000 (2.109)1 femtogram (fg) or 10-15 g ppq 3 attomoles or 3.10-18 2,000,000 (2.106)1 attogram (ag) or 10-18 g ppquint 3 zeptomoles or 3.10-21 2,000 (2.103)1 zeptogram (zg) or 10-21 g ppsext 3 yoktomoles or 3.10-24 2 (2.100)

1 yoktogram (yg) or 10-24 g ppsept Ghost mole 0

The most likely explanation for this is that very few ions actually reach the detector, hence, the ion statistics cause the measured isotopic ratio to fail normal threshold of +/- 20% from theoretical value

Detector Counts Human serum level 2378-TCDD ~ 1pg/g fat

0 5% fat 2g sample size = 10fg 2378-TCDD0.5% fat, 2g sample size 10fg 2378 TCDD= 2 107 molecules

50% 1 107 l l 50% recovery rate 1 107 molecules 1µl injected from 5µl 2 106 molecules Ionization efficiency 10% 2 105 molecules% fragmentation 10% 2 104 molecules% fragmentation 10% 2 10 molecules% transmit. HRMS 10% 2 103 molecules 5 l k 4 102 l l 5 cycles per peak 4 102 molecules 10ms dwell time/40ms 100 molecules…

Challenges (II) ‘Weight effect’…

Specie Mass Required Ion Isotope Level SpecieSpecie Mass Required Ion Isotope Level Specie resolution in cluster abundance (pg/μl) ratio

12C-2378-TCDD 321,8930 M+2 100% 0,05 1>8000

13C 2378 TCDF 321 9325 M+6 10% 50 100C-2378-TCDF 321,9325 M+6 10% 50 100

12C-2378-TCDD 319,8960 M 75% 0,05 1>8000

13C 2378 TCDF 319 9354 M 4 50% 50 65013C-2378-TCDF 319,9354 M+4 50% 50 650

[I i i i h l b i f d h ld

Challenges (III)[It is interesting that laboratories from around the world are

experiencing the same background problems arising fromthese ubiquitous contaminants It appears that the widespreadthese ubiquitous contaminants. It appears that the widespreaduse and release of PCBs have resulted in their unwanted andpersistent presence in the environment.

It is ironic that the advances in technology that haveallowed the progressive lowering of detection limits havereached a limit imposed by the very contaminants thetechnology was designed to measure.

Nevertheless, this background contamination poses aserious problem in terms of defining the limit of detection and

ifi i b b k d h b li bl d d

Ferrario et al., Chemosphere, 1997, 34, 2451

quantification above background that can be reliably detectedfor various samples matrices.]

Where we are Robust hardware makes the ‘sensitivity

enhancement dream’ come true Valuable coupling (2D not even considered yet) Implementation of quantification under QA/QC

Where we go Dedicated alternative sample prep. Blank level issues…(factor of 2-10 for MDL…)( ) Investigation of blood spots for screening

Take Home MessageCost Target Comprehensive iLODs NCI Robustness ID

GC‐HRMS ++++ +++ ‐ <fg ‐ +++ ++++GC‐QISTMS/MS ++ +++ ‐ >fg ‐ + +++GCxGC‐qMS ++ (+++) ++ <fg +++ (++) Low Cl ?GCxGC‐TOFMS ++++ ++ ++++ >fg ‐ +++ +++GCXGC‐HRMS ++++ (++++) ‐ ~ag ‐ ++ +GCxGC‐HRTOFMS +++ +++ +++ <fg ? (++) ?  ? ++++RBAs ++ + (++++) ? >fg ‐ ++ ‐