Agilent HPLC-ICP-MS Quick Start Guide

Application Note

Food and Environmental

Automated Sequencing of Elemental
Speciation Methods Using HPLC-ICP-MS
with a Quick Change Valve Head

Multi-element speciation analysis with unattended
switching of columns and mobile phases

Authors

Aimei Zou1
Shuofei Dong2
Yuhong Chen
Chee Sian Gan

1

Agilent Technologies, Inc.

Singapore

2

Agilent Technologies

(China) Co., Ltd.

2

1

Introduction

ICP-MS is the technique of choice for trace multi-element analysis in many industries
and sample types. But the mobility, bio-availability, and toxicity of several elements,
including As, Hg, Cr, Se, Br, I, Sn, Pb, and Sb, depends on the element’s chemical
form or species (1–6). Because of these differences, speciation analysis—where the
different chemical forms are separated chromatographically before measurement
by ICP-MS—may be required to give a complete assessment of risk. Speciation
analysis is especially important for samples where safety is of concern, such as
foods, environmental samples, and consumer goods.

To ensure the safety of consumers and the environment, many industries and
products are subject to regulations or guidance relating to the speciation of
elements. For example, in the case of arsenic, the World Health Organization (WHO)
recommends a maximum content of inorganic arsenic (iAs) of 10 µg/kg (ppb)
in food and drinking water (2). The European Union (EU) and United States Food

and Drug Administration (US FDA) have each published the
maximum levels of iAs in fruit juice and rice products. In
August 2020, the FDA finalized their guidance for industry
on iAs in rice cereals for infants, setting the “action level”
limit at 100 μg/kg (ppb) iAs (3). Mercury species, particularly
methylmercury, are subject to legislation in many countries,
including China, Japan, and Philippines. The WHO specifies

a maximum guideline concentration of 10 μg/L for bromate

in drinking water (2). There are also regulations in place to
control the level of hexavalent chromium (Cr(VI)) in products
such as drinking water, cement, leather, and children toys.
Cr(VI) is hazardous, while Cr(III) is an essential nutrient,
so speciation analysis is necessary to determine the
concentrations of the different forms to ensure compliance
with regulations.

To meet current and potential future regulatory requirements,
laboratories need fast, automated, reliable, and flexible
methods for the determination of various species of multiple
elements. High performance liquid chromatography (HPLC)
connected to ICP-MS is the most widely used technique
for elemental speciation analysis due to its selectivity and
sensitivity (4–10 ). Agilent ICP-MS and ICP-QQQ instruments
easily link with Agilent HPLC systems through optimized
interfaces and integrated software control (7). The
coupled systems are controlled from the Agilent ICP-MS
MassHunter software, simplifying the workflow from method
development, data acquisition, and data reporting.

HPLC-ICP-MS methods are typically used to detect species
that can be separated on a single column, which often
means they are limited to measuring the different forms
of a single element. However, labs often need to perform
speciation analysis of several elements in each sample, such
as monitoring As, Sn, and Hg species in seafoods. Some
different element species can be measured successfully
using the same column and mobile phase, but this approach
may lead to compromised conditions and degraded
performance. Labs may also need to run multiple speciation
methods on different sample types or assess multiple
columns and mobile phases during method development.
In these cases, automating a sequence to switch between
different speciation methods using an HPLC-ICP-MS system
fitted with a Quick Change valve can increase productivity
and add flexibility, while also allowing unattended overnight
operation. Agilent offers a range of column selector valve
heads that can switch between 2, 4, 6, and 8 column
positions. A solvent selection valve is also available, to
accommodate switching between up to 12 different mobile
phases.

Benefits of integrated, HPLC-ICP-MS with a Quick Change
valve head include:

• Automated sequencing of multiple speciation methods
using different columns and mobile phases. This
approach saves time, reagents, and cost, as well as
reducing errors by minimizing sample handling.

• Independent control of up to eight speciation methods
with customized queue order provides flexibility in
operation. All methods can be run in one unattended
sequence utilizing automated switching of columns and
solvents.

• Support for method development, especially to identify
the best column or mobile phase for the separation of
target species. A routine analysis can be run in sequence
with methods that are under development.

In this study, tests were run to assess the flexibility,
performance, and stability of the HPLC-ICP-MS system with
a Quick Change valve head for the automatic sequencing
of three separate speciation methods. The HPLC-ICP-MS
system was used for the measurement of inorganic arsenic,
methylmercury, and bromine and iodine species using
methods developed in previous studies (8–10 ).

Experimental

Chemicals and reagents

All chemicals and reagents used in this study were bought
from Sigma-Aldrich. A 1000 ppm stock solution of As(III) and
As(V) was prepared by dissolving the appropriate amount
of sodium arsenite and sodium arsenate (ACS grade, purity
>99%) into 1% HNO3. Methylmercury(II) (chloride) (ACS
grade, purity >99.5%) was used to prepare a 100 ppm stock
solution. Separate solutions of bromide, bromate, iodide, and
iodate were prepared from sodium bromide, sodium bromate,
potassium iodide, and potassium iodate, respectively (ACS
grade, purity >99%). The appropriate amount of each halide salt

was dissolved in Milli-Q de-ionized water (DIW, 18.2 MΩ•cm), to

give 1000 ppm stock solutions for each target species. Details
of the mobile phases used for the three analytical methods and
a column flush method are given in Table 2.

Standard preparation

Three calibration levels were prepared for each set of
elemental species at the concentrations given in Table 1,
by diluting the respective stock solutions with DIW.

2

Table 1. Calibration standard levels prepared for As(III), As(V), MeHg(II), Br-,

-

BrO

, I-, and IO

3

Calibration

Level

1 0.5/0.5 0.25 0.5/0.5/0.25/0.125

2 1.0/1.0 0.5 1.0/1.0/0.5/0.25

3 2.0/2.0 1.0 2.0/2.0/1.0/0.5

-

.

3

Mixture of

As(III)/As(V) (ppb)

MeHg(II)

(ppb)

Br-/BrO

Mixture of

-/I-

/IO

3

3

-

(ppb)

Instrumentation

For this work, an Agilent 1260 Infinity II LC system with
a quaternary pump and InfinityLab Quick Change
4-column-selector valve head (p/n G4237A) was used. The
HPLC was coupled to an Agilent 7900 ICP-MS using the
Agilent LC connection kit (p/n G1833-65200). A schematic
of the instrumentation is shown in Figure 1. The appropriate
HPLC column and mobile phase for each speciation method
were selected and changed automatically during the
sequence via the Agilent ICP-MS MassHunter software.

The quaternary pump can handle up to four mobile phases,
which was sufficient for the seven species (four elements)
measured in this study. For analysis requiring more than four
mobile phases, a 12 position/13 port solvent selection valve
(p/n G4235A) could be added. Similarly, when labs need to
run methods using up to eight different columns, an optional
Quick Change 8-column-selector valve head (p/n G4239C)
can be used in place of the 4-column-selector valve.

Multi-sequence acquisition method

Three analytical methods separated by wash steps were
included in this multimethod speciation sequence, as shown
in Table 2. Valve ports 1 to 3 were connected to the respective
speciation method columns, while valve port 4 was used to
flush out the system using DIW. HPLC and ICP-MS operating
conditions and parameters for each of the three speciation
methods are given elsewhere (8–10 ). As well as the different
elements’ acquisition masses, appropriate ICP-MS operating
conditions were applied for each HPLC method.

Table 2. Details of the separate methods used in the multimethod acquisition
sequence.

Method

Description

Speciation 1

Speciation 2

Speciation 3

Wash 4

Detail Valve

Speciation

of inorganic

As (As(III)

and As(V))

Speciation

of MeHg(II)

(CH3Hg)

Speciation

of bromide,

bromate,

iodide, and

iodate

Flushing

of HPLC

system

Position

1

2

3

4

Mobile

Phase

2.0 mM PBS/0.2 mM
EDTA/10 mM

CH3COONa/3.0 mM

NaNO3/2% ethanol,

pH 11.0 adjusted with

NaOH

Run time: 10 mins

2% methanol /0.5g/L

L-Cysteine, pH 2.3
adjusted with HCl

Run time: 4 mins

5.0 mM NaH2PO4/

15.0 mM Na2SO4 /

5.0 mM EDTA

Run time: 8 mins

DIW

Run time: 10 mins

Column

Anion

exchange

column,

Agilent p/n

G3288-80000

ZORBAX RRHT

Eclipse

Plus C18,

Agilent p/n

959941-902

Anion

exchange

column,

Agilent p/n

G3268-80001

No column

required

Figure 1. Schematic of the HPLC-ICP-MS system with Quick Change valve
head used in this study. The 4-column-selector valve head and quaternary pump
enabled automated selection of the different HPLC columns and solvents.

To demonstrate the flexibility of automated column switching
for routine unattended analysis, the three speciation methods
were run with a different sequence order over three days, as
shown in Figure 2. At the beginning of each day’s sequence
and after each speciation method, the wash method flushed
the HPLC-ICP-MS for 10 minutes using a continuous flow
of DIW. For compatible columns and mobile phases, the
separate wash method could be eliminated, with blank
injections at the start of each batch ensuring stabilization
under the new method conditions. Method performance
criteria such as linearity, recovery, and precision for the
speciation analysis of arsenic, mercury, bromine, and iodine
were evaluated over three days.

3

Figure 2. Experimental flowchart for the automated multi-method
HPLC-ICP-MS sequences run over three days.

Results and discussion

The performance of the HPLC-ICP-MS system with Quick
Change valve head was assessed based on the workflow
shown in Figure 2. On each of the three days, the three analytical
method batches were run in a different order, interspersed with
the wash method. Each method batch consisted of three
injections of each of the three calibration levels, plus four
injections of the blank, giving 39 injections for the three batches.
The addition of the four interspersed wash injections indicated
in Figure 2 gave 43 injections in total for each day’s sequence.
Each sequence was completed within 5.5 hours, regardless
of the different method run order.

Using the sequence on day 2 as an example, Figure 3
outlines the automated procedure and queue order that
was controlled by the ICP-MS MassHunter software.
The illustration in Figure 3 shows how the Quick Change
valve-based HPLC-ICP-MS system can be set up for routine,
unattended analysis of multiple batches in a user-defined
order. Running the different methods sequentially on one
HPLC system significantly improves the productivity and
turnaround times of speciation analysis, compared to running
the methods individually with manual column changeover.
The flexibility of the valve-based HPLC-ICP-MS system means
that up to eight speciation methods can be run sequentially
on one instrument, according to the requirements of the
laboratory.

Linearity of calibration

Calibration was carried out by measuring standard solutions

-

of As(III) and As(V), CH3Hg, and Br-, BrO

3

, I-, IO

-

in three

3

different batches over three days. Triplicate injections were
measured for each calibration level on each day. Excellent
linearity was observed for all analytes over the three days.
The representative calibration curves for all species obtained
on day 1 show excellent linearity, with R greater than 0.9995
(Figure 4).

Figure 4. Calibration curves showing excellent linearity. Top: As (III), As(V), and CH3Hg; Middle: IO

4

−

and I−; Bottom: BrO

3

−

and Br−.

3

Figure 3. Schematic showing the automated HPLC-ICP-MS procedure controlled by ICP-MS MassHunter software.

5

Figure 5. Overlaid chromatograms of A: inorganic arsenic, B: methylmercury, and C: bromine and iodine species obtained from nine injections of level-3 calibration
standards. Shows excellent stability of retention times and peak areas over three days. n=9 for each chromatogram.

Day to day stability of measured elemental species

Figure 5 shows overlaid chromatograms of the nine injections
of the level-3 calibration standard for iAs, CH3Hg, IO

−

BrO

, and Br− measured over three days. Within each day’s

3

−

, I−,

3

sequence, all species were separated and measured using
the optimum column and mobile phase conditions, switched
automatically during the run. The excellent reproducibility over
three days demonstrates the stability and applicability of the
multibatch HPLC-ICP-MS method with Quick Change valve.

Recovery of standard solutions

The recoveries of all elemental species were based on the
ratio of the calculated concentration from the calibration
curves relative to the expected concentration at the three
concentration levels. The recoveries for all species measured
over three days were within 90–110%, as shown in Figure 6.
The results confirm the stability and accuracy of the data
produced by the Quick Change valve-based HPLC-ICP-MS
system.

Precision

Method precision was calculated using within-day (intraday)
repeatability (RSDr) and between-day (interday) reproducibility
(RSDiR) based on the variation of peak area measurements of
the three repeat injections of each calibration level. Intraday
repeatability, denoted RSDr was determined by calculating the
percent relative standard deviation (%RSD) of peak area using
three injections of each calibration standard performed on
the same day. The RSDr values for all species were ≤ 8%.

Interday reproducibility, denoted RSDiR, was measured as %RSD
of peak area from a total of nine injections per concentration
level measured over three days. RSDiR values for all elemental

species were ≤10%, as shown in Table 3. The between-day

reproducibility was not affected by the queue order of the
speciation methods, which demonstrates the robustness of
the automatic valve-based HPLC-ICP-MS method.

Table 3. Between-day reproducibility of peak area for all elemental species
measured at three concentration levels.

Calibration

Level

Cal 1 2 6 7 5 4 3 5

Cal 2 4 6 9 8 6 10 8

Cal 3 3 4 7 5 5 5 4

As(III) As(V) CH3Hg IO

RSDiR of Peak Area (%)

-

BrO

3

-

-

Br

3

-

I

Figure 6. Recovery values for all elemental species measured at three

concentrations over three days.

6

Carryover assessment

The effectiveness of DIW to remove any residue or
contaminants from the LC flow path after each speciation
method was evaluated. The chromatograms in Figure 7,
which were obtained during three separate wash-method
runs on day 2, show that carry over between the different
speciation methods was reduced to acceptable levels of
<2% in each run.

Rinse run after MeHg speciation method.

Rinse run after Br/I speciation method. Rinse run after iAs speciation method.

Figure 7. Background signals in wash methods confirm minimal carryover between analytical method runs on day 2.

Conclusion

Multiple speciation analysis methods requiring different
columns and mobile phases were run automatically using an
Agilent 1260 Infinity II LC coupled to an Agilent 7900 ICP-MS.
The HPLC was fitted with an Agilent Quick Change column
selector valve head, which eliminated the need to manually
disconnect and reconnect different columns.

Three speciation methods were run enabling the measurement

-

of As(III) and As(V); CH3Hg; and Br-, BrO

3

, I-, IO

-

. ICP-MS

3

MassHunter was used to control the column selector valve,
selecting the queue order of the three speciation methods
and the wash cycle through the automatic positioning of
the appropriate column. Good levels of precision, recovery,
repeatability, and reproducibility were achieved for the
multiple speciation methods run each day, and using
different queue orders of the methods over three days.

Once an analyst has set up the columns, prepared the mobile
phases, and loaded the samples into the autosampler, the
HPLC-ICP-MS with Quick Change valve head can run up to
eight speciation methods. Multiple speciation methods can be
applied sequentially to the same samples, or different sample
batches can be analyzed sequentially using different methods.
For applications where multi-element speciation is of interest,
automating switching between different methods improves
productivity, shortens turnaround time, and reduces running
costs—important considerations for many laboratories.

References

1. P. Apostoli et al., Elemental speciation in human health
risk assessment, World Health Organization 2006,
accessed November 2020, http://www.inchem.org/

documents/ehc/ehc/ehc234.pdf

2. Guidelines for drinking-water quality: fourth edition
incorporating the first addendum. Geneva: World Health
Organization; 2017. License: CC BY-NC-SA 3.0 IGO

3. US FDA, Guidance for Industry: Action Level for Inorganic
Arsenic in Rice Cereals for Infants, accessed November
2020, https://www.fda.gov/regulatory-information/

search-fda-guidance-documents/guidance-industry­action-level-inorganic-arsenic-rice-cereals-infants

4. K. L. Ackley, J. A. Caruso, Separation Techniques, in:
R. Cornelis, J. Caruso, H. Crews, K. Heumann (Eds.),
Handbook of Elemental Speciation: Techniques and
Methodology, Wiley, England, 2003.

5. C. A. Ponce de León, M. Montes-Bayón, J. A. Caruso,
Elemental Speciation by Chromatographic Separation
with Inductively Coupled Plasma Mass Spectrometry
Detection, J. Chromatogr. A 974, 2002, 1–21.

6. M. Marcinkowska, D. Barałkiewicz, Multielemental
Speciation Analysis by Advanced Hyphenated Technique
– HPLC/ICP-MS: A review, Talanta , 161, 2016, 177–204.

7. Handbook of Hyphenated ICP-MS Applications, Agilent
publication, 5990-9473EN

8. M. Tanoshima, T. Sakai, E. McCurdy, Arsenic Speciation
Analysis in Apple Juice Using HPLC-ICP-MS with the
Agilent 8800 ICP-QQQ, Agilent publication, 5991-0622EN

9. S. Sannac, Y.H. Chen, R. Wahlen, E. McCurdy, Benefits of
HPLC-ICP-MS Coupling for Mercury Speciation in Food,
Agilent publication, 5991-0066EN

10. J. Nelson, S.F. Dong, M. Yamanaka, Simultaneous Iodine
and Bromine Speciation Analysis of Infant Formula by
HPLC-ICP-MS: Determination of four halogen species in
less than 6.5 minutes, Agilent publication, 5994-0843EN

www.agilent.com/chem

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This information is subject to change without notice.

© Agilent Technologies, Inc. 2021
Printed in the USA, January 22, 2021
5994-2943EN