Inorganic arsenic in rice using ICP-QQQMS with and without chromatography

Jörg Feldmann j.feldmann@abdn.ac.uk

Trace Element Speciation Laboratory (TESLA) University of Aberdeen

Scotland, UK

Webinar Agilent May 2014

Introduction into arsenic in rice HPLC-ICPMS: method description and results HG-ICPMS: method development and results

Comparison HG-ICPMS/HPLC-ICPMS

Table of content

“…that rice has higher inorganic arsenic concentrations than most other foods, and consequently, diets that rely heavily on

rice may contain the most inorganic arsenic.”

Schoof RA, et al. Fd Chem Toxicol 1999; 37:839-46.

A market basket survey of inorganic arsenic in food.

Arsenic in foodstuff

< 10 ng/g 100 ng/g 1000 ng/g

20,000-200,000

500-1,000

>500,000

1-2

50-90

5-10

220-1,100

1,900

Total Arsenic

Toxicity of arsenic

• Arsenic is a class I carcinogen

– Legislation (WHO, US-EPA, EU,…) : 0.010 mg/L in drinking water

– Food: no regulation, why…

Classes of arsenic species in food commodities

OH

AsOH O

OH

CH3

AsCH3

O

OH

OH

AsOH

OH

CH3

As+CH3

CH3

CH3

Inorganic As (iAs) Methylated arsenic (DMA, Tetra)

CH3

As+

CH3

CH3

CH2

C OH

O

O

CH3 CH3

As

CH3

CH3

O

O OH

OH

Arsenobetaine (AsB) Arsenosugars (AsSugar-OH, -SO4.)

GLU

CYS

GLUCYS

CYS

GLU

AsS S

S

GLY

Arseno-phytochelatins (AsPC3) Arsenolipids (AsHC, AsPL, AsFA)

OAs

CH3

CH3

OH

O

J. Feldmann & EM Krupp: Anal Bioanal Chem (2011), 399, 1735-1741.

Food control: Simplification of Speciation analysis by determining only three fractions of arsenic

1%

1%

18%

78%

2%

98%

Fish Molluscs

Seaweed

69%

1% 1%

1%

28%30%

Rice

toxic (iAs)

non-toxic (AsB)

potentially toxic

arsenic fractions

fat-soluble As

arsenosugars

other organoarsenicals

potentially toxic

2%

85%

5%

1%

7%

13%

5%

50%

13%

23%

9%

45%1 mg/kg

100 mg/kg

3 mg/kg

0.3 mg/kg

Tabloid study with trigger value Risk group: toddler and babies

• Babies have 3 times intake per body weight as

adults mg/kg/d and • Many baby products are based on rice 6 of 17 products could not be exported to

China ( ML: 0.150 mg iAs / kg).

AA Meharg, GX Sun, PN Williams, E Adomako, C Deacon, YG Zhu, J Feldmann, A Raab Environ. Poll. (2008), 152, 746-749

1989 – WHO: PTWI 0.015 mg kg-1 b.w.

1998 – Drinking Water Directive (EU) 98/83/EC (10 µg L-1).

2001 – The Environmental Protection Agency (EPA, U.S.) adopts a new arsenic in drinking water standard (10 µg L-1).

2005 – MLs of iAs and totAs in China for different food commodities (e.g. rice, 0.150 mg kg-1)

2008 – Paper on iAs levels in baby rice published.

2009 – FSA (UK) guidelines recommending no rice milk for young children, similar guidelines given in Denmark.

2009 – EFSA opinion paper on arsenic.

2010 – PTWI of 0.015 mg kg-1 b.w. from 1989 (WHO) was withdrawn by the Joint FAO/WHO committee.

2010 -2011 – Several Proficiency tests by IMEP in food and feed.

2011 – MLs on iAs in food in Australia.

2012 – Proposed draft MLs of iAs in rice by FAO/WHO.

2013 – Rice CRM (ERM - BC211), certified for iAs.

Food regulation – Selected events

…The inorganic forms of arsenic are more toxic as compared to the organic arsenic but so far most of the occurrence data in food collected in the framework of official food control are still reported as total arsenic without differentiating the various arsenic species….

…15 European countries > 100,000 results of As in food - 2/3 < LOD, - only 2 % speciation information

…The highest total arsenic levels were measured in the following food commodities: fish and seafood, … high concentrations in rice grains and rice-based products, …

….. In order to refine risk assessment of inorganic arsenic there is a need to produce speciation data for different food commodities to support dietary exposure assessment and dose-response data for the possible health effects….

As-speciation in rice is simple

OH

OH

As

OH

OH

OH

AsOH

O

OH

OH

AsCH3

O

OH

CH3

AsCH3

O

As(III)

As(V)

MMA

DMA

(CH3)4As+ TETRA (HR Hansen et al. J. Environm. Monit. (2011)

iAs

Introduction into arsenic in rice HPLC-ICPMS: method description and results HG-ICPMS: method development and results

Comparison HG-ICPMS/HPLC-ICPMS

Table of content

Extraction of rice for inorganic arsenic

200 mg rice + 1 % (v/v) HNO3 + 1 % (v/v) H2O2 Microwave oven (Mars5, CEM UK) in Corning tubes, - step 1: 5 min at 50oC, - step 2: 5 min at 75oC, - step 3: 30 min at 95oC. After cooling the samples were centrifuged.

Important for digestion and Oxidation of As(III) As(V)

Agilent 8800 QQQ (ICPMS)

HPLC-ICPMS

• SAX column: PRP X-100; 4.6 * 250 mm was used with 4 g/L (NH4)2CO3 buffer, 1 mL/min at RT; sample volume 50 L.

– Inorganic arsenic (sum of Asiii,Asv)

– Methylated arsenic (mainly DMA)

ICPMS HPLC

0

500

1000

1500

2000

2500

3000

3500

0 200 400 600 800 1000

Inte

nsi

ty

Time (s)

DMA

MMA

As(V)

What is the best method to detect arsenic with an ICPMS?

Arsenic detection in QQQ

As+ ArCl+

Zr+ Co+

O2

As+ AsO+ ArCl+

Zr+ ZrO+ Co+ CoO2

+

AsO+

Zr+

CoO2+

Set to 91u

ICP-MS

Single quadrupole

As+ ArCl+

O2

As+ AsO+ ArCl+

AsO+

Set to 91u

ArCl+, As+.

Set to 75u

Zr+, Co+

ICP-QQQ

Triple quadrupole

removes interfering polyatomics such as ArCl+, but the detecting m/z can still be interfered by newly generated polyatomics or other elements

Removes possible interring elements with Q1 and then removes interfering polyatomics such as ArCl+,

m/z 75: As, 40Ar35Cl

Arsenic detection in QQQ

M1 M2

Cell Q1

Set to M1

Q2

Set to M2

No gas Mode Q1: m/z 75 Q2: m/z 75 75As+

Oxygen mode Q1: m/z 75 Q2: m/z 91 75As16O+

Hydrogen mode Q1: m/z 75 Q2: m/z 75 75As+

Helium mode Q1: m/z 75 Q2: m/z 75 75As+

Ammonia mode Q1: m/z 75 Q2: m/z 91 75As(NH2)+

Q2: m/z 125 75As(NH3)2(NH2)+

O2, He, H2, NH3

ICP-QQQ settings

No gas H2 He O2 H2 + O2 NH3

Gas flow setting

- 2.4 ml/min 3.6 ml/min 30 % 3 mL/min + 30 %

2 mL/min (He) + 28 %

Energy Discrimination

(V)

5.0 5.0 1.0 -7.0 -6.0 -10.0

Wait time offset (ms)

0 0 0 2 2 5

MS-mode MSMS-mode

MS-mode MSMS-mode

MSMS-mode

MSMS-mode

m/z measured (Q2)

35, 75, 103

37, 75, 103 35, 75, 103 51, 75, 91, 103

37, 75, 91, 103

35, 75, 91, 103, 125

m/z 103 was used for the continuous internal standard Rh.

HPLC-ICP-QQQ of a rice extracts using different gas modes

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 2 4 6 8 10 12 14 16

rel. in

tensity (

off

-se

t)

retention time (min)

No gas

H2 6 mL

H2 7 mL

He

O2 (75 75)

O2 (75 91)

As(V) DMA

MMA ?

Check the As species eluting in the void: As(III) or (CH3)4As+

No gas mode or oxygen mode with mass transfer seems best suited

How good is the automatic integration using MassHunter for all gas modes ?

0

20

40

60

80

100

120

140

160

180

200

0 20 40 60 80 100 120 140 160 180 200

µg A

s / k

g r

ice m

anual..

µg As / kg rice MassHunter

H2 a

H2 b

He

no gas

O2 (75->75)

O2 (75->91)

Figures of merit No gas H2 He O2 H2 / O2 NH3

LoD (g/kg)

4.1 3.2 6.7 m/z 75: 3.3 m/z 91: 3.2

m/z 75: 6.6 m/z 91: 3.3

m/z 75: 12 m/z 91: 19

m/z 125: 54

BEC 0.15 0.098 0.099 m/z 75: 0.38 m/z 91: 0.10

m/z 75: 0.26 m/z 91: 0.040

m/z 75: 0.12 m/z 91: 0.32 m/z 125: 1.1

S/N 80 21 38 m/z 75: 10 m/z 91: 76

m/z 75: 35 m/z 91: 110

m/z 75: 15 m/z 91: 13 m/z 125: 4

Average RSD (%)

5.2 3.4 1.9 m/z 75: 6.0 m/z 91: 3.6

m/z 75: 2.9 m/z 91: 2.7

m/z 75: 5.8 m/z 91: 5.4

m/z 125: 7.8

Best method is either no gas or O2 or O2/H2 with As on m/z 91.

Optimisation of the gas modes (rice with high DMA)

All gas modes give the same result

0

20

40

60

80

100

120

140

160

180

200

no gas H2 He O2 (75) O2 (91) H2O2 (75) H2O2 (91) NH3 (75) NH3 (91) NH3 (125)

µg

As /

kg

ric

e

DMA

As(V)

NIST-1568 a

Optimisation of the gas modes (rice with high DMA)

0

20

40

60

80

100

120

140

no gas H2 He O2 (75) O2 (91) H2O2

(75)

H2O2

(91)

NH3

(75)

NH3

(91)

NH3

(125)

µg

As /

kg

ric

e

DMA

As(V)

concentration of the arsenic species control material, n=3

All gas modes give the same result.

No significant interference, hence any ICP-MS can be used.

HPLC-ICPMS analysis of CRM rice samples (n=4)

DMA MMA As(V)

BCR-211 (n=4)

119 +/- 12 15 +/-4.0 120 +/- 6.8

BCR-211 (certified)

119 +/- 13 124 +/- 11

NIST-1568a (n=2)

201 +/- 5.8 7.5 +/- 2.3 113 +/- 2.1

Literature 143–185 80–109

Summary of available methods for the analysis of arsenic in rice from various

countries

HPLC-ICPMS / HG-AFS

HPLC-ICPMS HPLC-ICPMS

HPLC-ICPMS

HPLC-ICPMS

ICPMS

Table taken from [Codex Committee on Contaminants in Food – March 2012 Report]

Analytical challenges with new regulatory guideline

Although HPLC-ICPMS can be used for speciation faster and cheaper and provide speciation information

Any intelligent fool can make things bigger, more complex, and more violent.

It takes a touch of genius -and a lot of courage - to move in the opposite direction.

- (Albert Einstein)

… if you are not a genius just look up old papers….

Introducing a selective step for iAs

– Selective volatilisation using hydride generation (NaBH4): • As(=O)(OH)3 (iAs) AsH3 (-55oC)

• (CH3)As(=O)(OH)2 (MMA) (CH3)AsH2 (+2oC)

• (CH3)2As(=O)OH (DMA) (CH3)2AsH (+35oC)

• AsB, As-sugars, As-lipids no volatile product !

…and reactions show pH dependence!

Selective determination of iAs without chromatography

S. Musil, AH Petursdottir, H Gunnlaugsdottir, EM Krupp, J Feldmann

Analytical Chemistry (2014) 86, 993-999

Standard Agilent generator in CF mode

Vmixer = 0.23 ml

vcarrier gas = 0.3 – 0.4 l min-1 ; Makeup gas introduced downstream the GLS

vNaBH4 = 0.5 ml min-1

vsample = 0.5 ml min-1

vHCl = 2.5 ml min-1

2% NaBH4 in 0.1 M NaOH

High concentration of HCl

HG-ICPMS (wet/dry plasma)

WET plasma

DRY plasma

S. Musil, AH Petursdottir, H Gunnlaugsdottir, EM Krupp, J Feldmann

Analytical Chemistry (2014) 86, 993-999

Hyphenation of HG to ICP-MS(MS) – WET plasma conditions

Influence of HCl in the reaction mixture

5M HCl was chosen to eliminate DMA signal satisfactorily!!

MMA sensitivity 45.4 ± 0.2% of iAs(V) sensitivity

DMA sensitivity 2.2 ± 0.05% of iAs(V) sensitivity

0

100000

200000

300000

400000

500000

600000

700000

800000

0 5 10

Sig

nal in

ten

sit

y (

CP

S)

Concentration of HCl for HG generation (M)

Dependence of signal of As species (1 ppb) on concentration of HCl, results corrected to IS 103Rh, signals corrected to blank

iAs(V)

MAs(V)

DMAs(V)

iAs(V) pre-reduced byKI/ascorbicacid and HCl

S. Musil, AH Petursdottir, H Gunnlaugsdottir, EM Krupp, J Feldmann

Analytical Chemistry (2014) 86, 993-999

Rice samples

Speciation for iAs without chromatography

Rice samplea

HG- ICP-MS

HPLC- ICP-MS

Total As

(certified)

iAs

(reported)

JRC 203 92 ± 4

101 ± 5

JRC 165 93 ± 4

103 ± 3

NIST 1568a

100 ± 6

94 ± 5

290 ±

30

94 ± 12

Concentration of iAs in rice samples (μg kg–1) found:

and HG-ICP-MS three times faster than HPLC-ICP-MS

Samples

from

PT (2011)

S. Musil, AH Petursdottir, H Gunnlaugsdottir, EM Krupp, J Feldmann

Analytical Chemistry (2014) 86, 993-999

Oxygen vs no gas mode using HG-ICPMS

AH Petursdottir, N Friedrich, S Musil, A Raab, H Gunnlaugsdottir, EM Krupp, J Feldmann, Analytical Methods (2014) doi:10.1039/C4AY00423J

No gas mode and oxygen mode show the same results! no ArCl interference despite the high HCL concentration. All ICP-MS models can be used for this application.

Can the results be confirmed for rice with different DMA/iAs ratios?

Samples

• 32 rice samples, bought in local stores in Aberdeen, UK.

• 12 rice samples, grown under arsenic exposure.

Extraction

• Microwave extraction, 1% HNO3 and 1% H2O2.

AH Petursdottir, N Friedrich, S Musil, A Raab, H Gunnlaugsdottir, EM Krupp, J Feldmann, Analytical Methods (2014) doi:10.1039/C4AY00423J

HG (iAs conc g kg-1 )

0 100 200 300 400 500

HP

LC

(iA

s c

onc m

g

g k

g-1

)

0

100

200

300

400

500

95% Confidence Band

95% Prediction Band

44 rice samples (n=3)

Slope = 0.97

AH Petursdottir, N Friedrich, S Musil, A Raab, H Gunnlaugsdottir, EM Krupp, J Feldmann, Analytical Methods (2014) doi:10.1039/C4AY00423J

Conclusions

• HPLC-ICP-QQQ is ideally placed for the determination of iAs in rice

• Every gas mode gives acceptable results highlights highest flexibility and 7700 or 7900 can also be used.

• HG-ICPMS is selective enough to determine only iAs

– HPLC not needed!

– Faster determination than using chromatography

– Straight forward data treatment • no peak integration

• easier SOP

Acknowledgement

• Asta Petursdottir

• Helga Gunnlaugsdottir

• Eva M Krupp

• Stanislav Musil

• Nils Friedrich

• Andrea Raab

• Jenny Nelson

and

• Cornelius Brombach for the artwork

Further events

• HPLC-ICP-QQQ use for sulfur speciation in combination with arsenic in garlic

• including the use of ESI-qTOF

75As 75As16O+

32S 32S16O+

watch that space…

BNASS/TraceSpec Aberdeen, Scotland,

31st Aug- 4th Sept. 2014

BNASS 2014 –

17th Biennial Nat. Atomic Spectros. Symp.

TraceSpec 2014 –

14th Workshop on Progress in Trace Metal

Speciation for Environmental Anal. Chem.

http://www.abdn.ac.uk/events/bnass-tracespec-2014/