developing robust high throughput methods with sub ppt

Thermo UK Water Seminar April 2016

Developing robust high

throughput methods with

sub ppt quantitation levels

in water using LC-QQQ, GC-

QQQ and ICP-MS

Paul Gribble – ALcontrol Laboratories


ALcontrol

Laboratories Eu ope s la gest i depe de t

testing laboratory

Presenter

Paul Gribble Technical Director

[email protected]


We strive to become the market leader in the environmental, food and oil analysis that supports

ou lie ts o plia e e ui e e ts a d p ote ts pu li health. Th ough ad a ed a alysis a d production processes and with the best staff, we aim to set a new service standard for our

industry and reward those who invest time and money working with ALcontrol.

Service Coverage

Mission

• Austria

• Belgium

• Denmark

• Finland

• France

• Germany

• Ireland

• Norway

• Portugal

• Sweden

• Spain

• Switzerland

• United Kingdom

• Kuwait

• Poland

• Russia

• Ivory Coast

• South Africa

• UAE

• With logistics

partners

• In-country service and

support

• Labs and service

centres

ALcontrol – Key information


1500

Building an international company ...

1992 ALcontrol acquired

by Yorkshire Water

as the foundation of

building an

international lab

network

Rapid growth in

Environmental testing

market driven by

contamination

concerns and new

legislation in the

Netherlands, other

European countries

and the US

1983

1977 ALcontrol formed

as subsidiary of

Proton Wilton in

Etten Leur, The

Netherlands

1998 Acquires Biochem

in Netherlands and

ACS in UK as

entrance into Food

testing market

1999 Acquires KM lab and Svelab

as bold move into Nordic

market and starts to

rationalise and streamline

these operations.

ALcontrol enters rapidly

growing soil testing market

in UK by acquisition of

Geochem

2003 ALcontrol expands into

emerging French and

Belgian soil testing

markets

2000 ALcontrol enters

rapidly growing soil

testing market in UK

by acquisition of

Geochem

2011 Acquisition of

Labnett in Norway

as momentum

completing

coverage of

Scandinavian

markets

2012 Opening of new

German office in

Frankfurt to open

up and develop

largest market in

Europe

1978 ALcontrol starts

testing

environmental soil &

groundwater with 3

employees

2008 ALcontrol

expands across

Øresund bridge to

become

challenger in

Danish Food and

Environment

market

2007 Invests in

coverage of

Spain and

Portugal to

extend European

Coverage

2005 ALcontrol

purchases

Robertson's Oil

testing and

invests in new

laboratory to

enable European

expansion

Employees

IT and

support

staff

Management

Senior

Scientific Staff

Professionally

Trained

Laboratory

Technicians


Environmental testing and advice • Full range of contaminated land, ground, surface and

waste water, air, asbestos and asphalt testing.

• Wide range of standard matrices with full range of testing

capability. Ability to handle high volume requirements.

• ALcontrol centres of excellence for dioxin, soil gas,

asbestos, saline analysis.

• Custom low level detection consulting and method

development.

• Coolchain and specialist logistics, pre-scheduling for

waste and monitoring contracts.

• Online ordering and reporting including data management

to wide range of export formats : AGS, SIKB, equis etc. • Servicing all 20 of the world’s top 20 environmental consulting organisations.

• Expert advisor to Government organisations

in scoping testing regime regulations.

• Accreditations and recognitions allowing

testing majority of countries in Europe,

Middle East and Africa.

• One stop shop for large scale and

multicountry projects including subcontractor

management, import licencing and logistics,

and geotechnical testing.


ALcontrol - a trust organisation

• ISO 17025 is at the centre of the ALcontrol

laboratory system; all of our labs are

accredited.

• As an active member of ILAC, many of

ALcontrol’s laboratories hold multiple

country recognition allowing us to deliver

specialised services compliant with local

rules.

• In addition ALcontrol holds many vendor

and customer specific certifications and

participates in many proficiency testing

schemes.

• We put Integrity First in our company values

and hold all our staff to high personal

standards.

• Where applicable we conduct ethics audits to

be compliant with customer schemes.

• We promote staff openness and encourage

comments and improvement observations,

including having a well-publicised whistleblower

line and a policy to encourage openness.


Legislative background to new method development



Water Framework Directive (2000/60/EC)

• Came into force 22nd December 2000

• Establishes a legal framework to protect and

restore clean water across Europe and ensure its

long-term, sustainable use.

• Based on River Basin Districts.

• Many River Basins cover more than a single

country.

• Where this is the case, cooperation between

member states is required to ensure the quality

of the river basin.

• For individual water bodies, the directive defines:

o Good Ecological status

o Good Chemical status



Priority Substances Directive (2013/39/EU)

• Good Chemical Status is defined by EQSs

• EQSs are now set in the Priority Substances

Directive

• 2013/39/EU updated and amended WFD

(2000/60/EC) and Directive on Environmental

Quality Standards (2008/105/EC)

• 2013/39/EU Must be transposed into national law no later than 14/09/2015

• A number of new substances have been added to the original 33,

taking the total to 45

• 21 of these are Priority Hazardous Substances

• Toxic

• Persistent

• Bioaccumulative


Three different Environmental Quality Standards (EQSs)

1. AA-EQS: Annual Average

2. MAC-EQS: Maximum Allowable Concentration

3. Biota – EQS: set primarily in fish, although some are in crustaceans and molluscs.

Where prescribed biota-EQS take precedence over water concentrations


LoD required (µg/l)

LoQ required (µg/l)

AA-EQS (µg/l)

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

NiPb

Cd

• QA/QC directive: LoQ

must not be higher

than 30% of EQS

• Deriving LoD and LoQ

from ISO13530 LoQ is

approximately

2.15xLOD

• LoD must be 1/7th AA-

EQS


EQS derivation • Is toxicity based

• Can be for either human

consumption or other high

order predator, which ever is

the most sensitive receptor

• Where there is a small data set

(few species or sampling

points) a large safety factor is

used

• This is to ensure coverage (as

far as possible) of all

potentially affected species


• The use of a large safety

factor drives down EQS

detection limits


Priority Substances listed in directive 2013/39/EU with

Priority Hazardous Substances in bold


Alachlor Hexachlorobenzene Trichlorobenzenes

Anthracene Hexchlorobutadiene Trichloromethane (chloroform)

Atrazine Hexachlorocyclohexane Trifluralin

Benzene Isoproturon Dicofol

Brominated diphenylethers Lead and its compounds

Perfluorooctane sulfonic acid and its

derivatives (PFOS)

Cadmium and its compounds Mercury and its compounds Quinoxyfen

Chloroalkanes, C10-13 Naphthalene Dioxins and dioxin-like compounds

Chlorfenvinphos Nickel and its compounds Aclonifen

Chlorpyrifos (Chlorpyrifos-

ethyl) Nonylphenols Bifenox

1,2-dichloroethane Octylphenols Cybutryne

Dichloromethane Pentachlorobenzene Cypermethrin

Di (2-ethylhexyl) phthalate

(DEHP) Pentachlorophenol Dichlorvos

Diuron Polyaromatic Hydrocarbons (PAH) Hexabromocyclododecanes (HBCDD)

Endosulfan Simazine Heptachlor and Heptachlor epoxide

Fluoranthene Tributyltin compounds Terbutryn


Priority (Hazardous) Substance metals and Specific Pollutant EQSs


AA-EQS

(µg/l)

MAC-EQS

(µg/l)

Biota

(µg/kg) Notes

Cadmium and its

compounds 0.08 - 0.25 0.45-1.5

PHS. EQS dependent on hardness

of water - softer water has lower EQS

Lead and its

compounds 1.2 14 Bioavailable fraction applies to AA-EQS

Nickel and its

compounds 4 34 Bioavailable fraction applies to AA-EQS

Mercury and its

compounds 0.07 20 PHS

Bioavailable metals in water are calculated using metal, DOC, calcium and pH result.

There are also specific pollutants including aluminium, chromium, copper, iron and

zinc.


Large scale monitoring programmes

In the UK, large scale sampling and analysis programmes have been done:

- National Demonstration Programme (NDP)

- Chemical Investigations Programme (CIP)

- Starting in April 2015 Chemical Investigations Programme 2 (CIP2)

- CIP1 looked at efficiency of

sewage works to remove

chemicals and also point

sources of chemicals

- One surprising conclusion

of CIP1 was that many

contaminants enter sewage

works via domestic rather

than industrial sewers

- This makes point source

control difficult


Chemical investigations Programme 2 (CIP2)

- The main phases of CIP2 are looking at:

- Dilution effects for PS/PHS and specific pollutants in rivers once discharged from

Sewage works

- River samples being taken from upstream of sewage works to monitor

background levels of contaminants

- Sewage samples taken from Final Effluent discharge

- River samples taken

downstream of discharge

- Modelling to be done to see

what dilution effect the river

courses are having

- CIP2 also has a smaller phase

looking at emerging

contaminants and how well

sewage works remove these

(pharmaceuticals and

oestrogen based steroids)


Metal (total and

dissolved) Required LOD µg/l

Nickel 0.5

Lead 0.2

Copper 0.3

Zinc 0.5

Cadmium 0.04 (0.02 in river)

Mercury 0.001

Iron 50

Aluminium 50

Chromium 0.5

CIP2 Metals and detection limit requirements


Ther o Scie tific™ X Series™ 2 ICP-MS, now old technology

⁻ 1st purchase 2003

⁻ 3x without cell

⁻ 1x with cell

⁻ Great workhorses

AA-EQS (µg/l) MAC-EQS (µg/l)

CIP2 LOD

(µg/l)

X Series 2 ICP-MS

LoD – dissolved

(µg/l)

Cadmium and its compounds 0.08 - 0.25 0.45-1.5 0.02 0.1

Lead and its compounds 1.2 14 0.2 0.02

Nickel and its compounds 4 34 0.5 0.15

Mercury and its compounds 0.07 0.001 0.15

Metals analysis current state


New technology – Ther o Scie tific™ iCAP™ Q ICP-MS

Future state


Agreed on the following

objectives

1. EQS and CIP2 level

analysis in river and

sewage

2. Routine analysis of

heavy matrix samples

3. All validated to

ISO17025 standard

The o S ie tifi ™ iCAP™ Q ICP-MS – start of a journey of development

Engaged with Thermo Fisher Scientific about what we

needed from a new instrument


Agreed configuration

1. iCAP Q ICP-MS with collision cell allowing single

gas usage (pure He)

2. CETAC ASX-520 autosampler with ASXpress Plus

rapid sample introduction.

3. ESI PFA-ST nebuliser

4. Peltier cooled, quartz cyclonic spray chamber.

5. Online internal standard kit for iCAP Q ICP-MS

The o S ie tifi ™ iCAP™ Q ICP-MS – start of a journey of development


1. High sensitivity

2. Highlighting of environmental issues

Initial successes


1. Low level background contamination

from zinc, noticeable because of the

additional sensitivity of the Thermo

S ie tifi ™ iCAP™ Q ICP-MS and looking

for 0.5µg/l zinc for CIP2

2. Looked at a wide range of possible

sources

3. Putting controls in place; we now have

background equivalents of c.0.5 µg/l

4. The main control measure is minimum

contact with any possible sources

Initial issues - Environmental


Minimisation of environmental issues

Initial successes


- Poor RSDs – fronting and tailing in loop

- Heavy matrix

- Poor recovery and divergence of ISs followed by

long stabilisation time

Robustness testing using high matrix samples

We have see the same

phe o e o o othe e do s ICP-MS. The issue is related to

technique and is not instrument

specific.


Initial issues – Instrument configuration

1. Flow stability

2. Flow control

─ Trialled different bore

loops on autosampler

─ Initial front end dilution

and tailing using wide

bore loop

─ Switch to very narrow

bore loop helped, but

gave pressure build up

─ Settled on mid-sized

bore loop as best

compromise


₋ Internal standard

being used to

create 4x dilution

₋ Achieved using

2x diameter bore

I/S tubing

compared to

carrier tubing

₋ Green-orange

tu i g . ” and black-black

. ” ID

Dilution with Internal Standard


Improved speed of recovery and better stabilisation with the

4x dilution on high matrix samples

Improved Internal Standard Recovery

Samples

Calibration

Washes


Some samples are really saline – sodium intensity readily visible

Salts were dropping out of solution as it was super saturated

How high matrix?

View from the in-built camera


1. Bore of the loop tubing varied

2. Pump tubing balance changed

3. Nebuliser switched from ESI PFA-ST to Burgener Miramist, as the

latter is designed to deal with higher dissolved solids samples

Configuration changes


Selenium mass selection based on cell ability and instrument sensitivity

₋ 80Se a t e used e ause of the 40Ar2 interference.

₋ 78Se is also interfered by Ar2 (38Ar40Ar) but to a much lower extent than 80Se.

₋ Collision cell mode easily attenuates these polyatomic interferences.

₋ Previously 82Se as used; it s o possi le to use 78Se for even greater sensitivity.

Selenium mass selection

Isotope Abundance Interference Interference Interference

74Se 0.9

76Se 9.0

77Se 7.6

78Se 23.6 38Ar40Ar 12C66Zn, 14N64Zn 156Ga++

80Se 49.7 40Ar 40Ar 40Ar40Ca

82Se 9.2 1H81Br 1H16O65Cu


The The o S ie tifi ™ iCAP™ Q ICP-MS allows switching by element

between cell and non-cell mode, but, like all quadrupole cell based

instruments, with an effect on the signal for low masses.

In cell mode, low masses show low sensitivity as they have low kinetic

energy and are strongly scattered by the cell gas (pure He)

Increased counts in standard (i.e. non-cell mode) allow for much lower

LOD for these lighter, non-interfered elements.

7 Lithium 9 Beryllium 11 Boron

Std Conc

(ppb) 100 100 10000

KED Mode

counts/s 5940 3411 238000

STD Mode

counts/s 1003000 233000 27100000

Is cell mode always best?


E e ith all the ha ges e e ade, e e still ot really at

a stage where the system is suitable for routine use with high

matrix samples.

This is not limited to the The o S ie tifi ™ iCAP™ Q ICP-MS

as we have trialled other systems with the same issues.

The answer would seem to be in front-end delivery and

dilution of the sample into this new generation of highly

sensitive ICP-MSs.

New generation ICP-MSs with heavy matrices - Summary


ESI prepFAST Auto-dilution System

• prepFAST is an evolution of the FAST valve

discrete sample introduction system

• The prepFAST is a dual FAST valve system:

– Samples are loaded on to the loop in

the first valve where syringe pumps

accurately dilute / add internal

standard to the sample

– The diluted sample is then passed to

the second loop for FAST valve transfer

to the The o S ie tifi ™ iCAP™ Q

ICP-MS

• Single standalone system, ICP-MS supplier

independent

• Fully integrated support for the prepFAST is

provided in the The o S ie tifi ™ Qteg a™ ISDS software that controls the iCAP Q

ICP-MS


ESI Inc prepFAST Auto-dilution using Qtegra ISDS

• Ther o Scie tific™ Qtegra™ ISDS

Software provides complete

software control of the prepFAST

• Automated prescriptive dilution for

preparation of:

– Samples

– Standards

• Automated intelligent dilution:

– Over Calibration Range

Autodilution

– Internal Standard Range

Autodilution


R2=0.99996

Standard Preparation using Auto-dilution

A single standard stock (Rack 3, Vial 2) is used

to generate a 10 point calibration (from 0.01

to pp ) ith DF s f o 1 to 400

- Dual stocks can be used to extend

automated calibration ranges even further


Advanced Auto-dilution: Calibration Over-Range

• The user defines:

• The maximum allowed over-range limit

as a percentage of the top calibration

standard, e.g. 110%

• The target concentration of the analyte

after auto-dilution, e.g. 60%

Everything else is done by Thermo

Scie tific™ Qtegra™ ISDS


Advanced Auto-dilution: Internal Standard Recovery

• The user defines:

– The range of acceptable Internal

Standard Recovery, e.g. 60 - 125%

– The autodilution factor step and number

of possible dilutions

Everything else is done by Thermo

Scie tific™ Qtegra™ ISDS


Recap: Performance with 4x on-line dilution, using a Y-piece

Improved Internal Standard Recovery with the ESI Inc prepFAST

Samples

Calibration

Washes


Improved Internal Standard Recovery with the ESI Inc prepFAST

Calibration Samples Method validation: DL blanks, cal checks

Internal Standard recovery now significantly more stable and

autodilution i gs a y outlie s auto ati ally i to a ge…

Performance with 40x prescriptive on-line dilution with the ESI Inc prepFAST


• A closer look at sample 24

• > 8 % NaCl in this sample!

• Required internal standard recovery 85-125%

• Prescriptive dilution (PD) at 40 fold

• Autodilution steps:

• 1 autodilution to 50 fold



After the 3rd dilution, the internal standard

recovery is within the specified range

analysis can continue

PD:40

AD:50

AD:60

AD:70

Improved Internal Standard Recovery and Intelligent Dilution with the ESI Inc prepFAST


Thermo ICAPq with ESI Inc prepFAST – routine operation

• Calibrations all prepared from a single standard on the

autosampler

• Running with routine 10x dilution

• Using automatic dilutions where internal standard

response is outside specification

• Using automatic dilutions where samples are over

calibration range

• Have MCERTS accreditation for Final Effluent, Crude

Sewage and Trade Effluent

• Have ISO17025 for a broad range of other matrices


Chemical investigations Programme 2 (CIP2)

- The main phases of CIP2 are looking at:

- Dilution effects for PS/PHS and specific pollutants in rivers once discharged from

Sewage works

- River samples being taken from upstream of sewage works to monitor

background levels of contaminants

- Sewage samples taken from Final Effluent discharge

- River samples taken

downstream of discharge

- Modelling to be done to see

what dilution effect the river

courses are having

- CIP2 also has a smaller phase

looking at emerging

contaminants and how well

sewage works remove these

(pharmaceuticals and

oestrogen based steroids)


Priority Substances listed in directive 2013/39/EU with

Priority Hazardous Substances in bold


Alachlor Hexachlorobenzene Trichlorobenzenes

Anthracene Hexchlorobutadiene Trichloromethane (chloroform)

Atrazine Hexachlorocyclohexane Trifluralin

Benzene Isoproturon Dicofol

Brominated diphenylethers Lead and its compounds

Perfluorooctane sulfonic acid and its

derivatives (PFOS)

Cadmium and its compounds Mercury and its compounds Quinoxyfen

Chloroalkanes, C10-13 Naphthalene Dioxins and dioxin-like compounds

Chlorfenvinphos Nickel and its compounds Aclonifen

Chlorpyrifos (Chlorpyrifos-

ethyl) Nonylphenols Bifenox

1,2-dichloroethane Octylphenols Cybutryne

Dichloromethane Pentachlorobenzene Cypermethrin

Di (2-ethylhexyl) phthalate

(DEHP) Pentachlorophenol Dichlorvos

Diuron Polyaromatic Hydrocarbons (PAH) Hexabromocyclododecanes (HBCDD)

Endosulfan Simazine Heptachlor and Heptachlor epoxide

Fluoranthene Tributyltin compounds Terbutryn


Possible GC-MSMS analytes

1. Brominated diphenylethers

2. Di (2-ethylhexyl) phthalate (DEHP)

3. Fluoranthene

4. Nonylphenols and Octylphenols

5. Polyaromatic Hydrocarbons (PAH)

6. Tributyltin compounds

7. Cypermethrin

8. Hexabromocyclododecanes (HBCDD)


Possible LC-MSMS analytes




4. Cypermethrin


6. Perfluorooctane sulfonic acid and its

derivatives (PFOS)


Analytes possible LC-MSMS or GC-MSMS




4. Cypermethrin



How do we decide which technique to use?

What do we do already?

Which technique do we feel most

comfortable with?

Do standard methods exist?

What does literature suggest?

What is the detection limit requirement?

What equipment do we have available?


Available Kit

TSQ 8000 Evo

TRACE 1310


Available Kit

TSQ Quantiva

with Ultimate

3000 HPLC


Analytes possible LC-MSMS or GC-MSMS




4. Cypermethrin



What is HBCDD

Brominated Flame Retardant

Isomeric

Slightly soluble 3.4μg/l


Development of HBCDD in water

What do we do already? We have no

experience of analysing HBCDD

Which technique do we feel most comfortable

with? GC

Do standard methods exist? Yes, ut…

What does literature suggest? Variations

between GC and LC methods

What is the detection limit requirement?

0.23 ng/l

What equipment do we have available?

GC and LC


Literature search for HBCDD analytical methods

Isomeric conversion occurs

Conversion may happen during GC analysis

GC a o ly p o ide total HBCDD esult

LC-MSMS provides specific isomer information

Different isomers may have different

instrument responses

Different environmental media have been

observed to have different isomer ratios

Predominance of α-HBCDD in aqueous

samples

Predominance of γ-HBCDD in sand eel

study


HBCDD development - Initial Thoughts

LC-MSMS probably give best opportunity to

quantify accurately because

Individual isomers are calibrated, which

should account for isomer specific

instrument responses

Isomer conversion not known to occur in LC

system

Isomer distribution in samples seen in

lite atu e, ut e do t k o if ou sa ples will behave the same way

All the above gives some future proofing


HBCDD development - Initial Observations

HBCDD does not provide a stable signal

Lack of stable signal Lack of robust analysis,

particularly statistical LOD

In full scan HBCDD different adducts are observed

Chorine, bromine and dimeric adducts observed

Cl-


HBCDD development – Initial Observations

Direct Infusion of Methanol standard:

Direct Infusion of Acetonitrile standard:

Direct infusion of

standards shows a

different spectra

for standards in

acetonitrile and

methanol


HBCDD development – Acting on observations

Can we promote adduct formation?

References suggest that ammonium chloride can be

used to form solely chlorine adducts

We see max 5:1 Chlorine adduct:native HBCDD

• Left -

Responses:

α<β<γ

• Right - In

samples, we

typically see:

α>β>γ


HBCDD development – Acting on observations

Dominant bromine adducts observed in

acetonitrile. HBCDD is not stable in ACN?

Can we form bromine or iodine adducts?

Yes, though they are more difficult to fragment

No overall improvement, so: back to chlorine

adducts

Optimised Ammonium Chloride concentration in

mobile phase

Adduct formation is reproducible

Calibration curve can be created, with good

repeatability


HBCDD development – Back to chlorine adducts

Note: HBCDD native signal is now stable, although lower

intensity than Cl-adduct

α-HBCDD Signal = 150000

R2 = 0.9931

α-HBCDD-Cl-adduct

Signal = 340000 R2 = 0.9992


HBCDD – Final configuration

Quantification based on Chlorine adduct

Calibration using individual isomers α, β and γ-

HBCDD

Individual 13C internal standards incorporated

which minimise:

Matrix effects

Extraction variability

Variable isomer abundances

Variable responses

Extraction is liquid-liquid


HBCDD – Proof the method works

CIP2 have commissioned a PT scheme for

compounds not offered by Aquacheck

So far there have been two rounds for HBCDD

Each round consists of 3x different samples

ALcontrol have satisfactory Z-scores (2z <= -2z)


Thermo – Neville Llewellyn and

Simon Nelms

Thanks

ALcontrol – Adam Gower, Kate

Rawlinson, Alex Murray, Joanna

Strzalkowska, Clare Sandman and

Chris Birtwistle


Questions?


For more information please contact:

[email protected]

developing robust high throughput methods with sub ppt

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