Manual of Methods of Analysis of Foods-

Metals and Minerals

 Table of Contents

S.No. Method No. Particulars Page No.

1. FSSAI 09.001:2024 Method for the analysis of trace elements in food 3-11
by Inductively Coupled Plasma-Optical
Emission Spectroscopy Using Microwave
Assisted Digestion
(ICP-OES)
2. FSSAI 09.002:2024 Method for Determination of Calcium, Copper, 12-18
Iron, Magnesium, Manganese, Potassium,
Phosphorus, Sodium, and Zinc in Fortified Food
Products by Inductively Coupled Plasma-Optical
Emission Spectrometry (ICP-OES)
3. FSSAI 09.003:2024 Method for the analysis of trace elements in 19-29
Water by Inductively Coupled Plasma Mass
Spectrometry (ICP-MS)
4. FSSAI 09.004:2024 Method for the analysis of Arsenic, Cadmium, 30-36
Mercury, and Lead in Foods by Pressure
Digestion and Inductively Coupled Plasma-Mass
Spectrometry (ICP-MS)
5. FSSAI 09.005:2024 Method for the analysis of Chromium, Selenium,
and Molybdenum in Infant Foods and Adult 37-40
Nutritional Food Products by Inductively
Coupled Plasma-Mass Spectrometry (ICP-MS)
6. FSSAI 09.006:2024 Method for the analysis of Total Iodine in Infant 41-46
Foods and Adult/Pediatric Nutritional foods by
Inductively Coupled Plasma–Mass Spectrometry
7. FSSAI 09.007:2024 Method for the Determination of Methyl 47-52
Mercury and Total Mercury in Seafood by High
Performance Liquid Chromatographic-
Inductively Coupled Plasma-Mass Spectrometry
(ICP-MS)

2
 Method for the analysis of trace elements in food by Inductively
Coupled Plasma-Optical Emission Spectroscopy
Using Microwave Assisted Digestion

Method No. FSSAI 09.001:2024 Revision No. & Date 0.0

Scope This method describes procedures for using inductively coupled plasma-
optical emission spectrometry (ICP-OES) for determination of total
element concentration (mass fraction) in food. The method was validated
with the following foods: milk, cheese, bacon, tuna, eggs, peanut butter,
corn, bread, pancakes, cereal, prune juice, lemonade, broccoli, sweet
potato, spaghetti & meatballs, mayonnaise, beer, beef baby food, haddock
and pears. Other matrices may be analyzed by these procedures if
performance is demonstrated for an applicable analyte in the matrix of
interest, at the concentration levels of interest.
It should be noted that aluminum results could be biased low in some
samples because of insoluble aluminum compounds especially if silica is
present. Thallium is listed conditionally because although fortification
recoveries were acceptable during method validation, no reference
materials were available.
Element LOD (mg/kg) LOQ (mg/kg)
Aluminum (Al) 0.8 2
Arsenic (As) 2 4
Barium (Ba) 0.05 2
Boron (B) 0.3 0.8
Cadmium (Cd) 0.3 0.9
Calcium(Ca) 8 30
Chromium (Cr) 2 5
Cobalt (Co) 0.3 0.8
Copper (Cu) 0.1 0.3
Iron (Fe) 0.2 0.3
Lead (Pb) 3 6
Magnesium (Mg) 2 6
Manganese (Mn) 0.2 0.4
Molybdenum (Mo) 0.4 1
Nikel 0.9 3
Phosphorus (P) 2 6
Potassium (K) 20 40
Sodium (Na) 2 5

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 Strontium (Sr) 0.03 0.07
Thallium (Tl) 2 6
Vanadium (V) 0.2 0.5
Zinc (Zn) 0.3 0.5

Aluminum concentrations using the method do not account for aluminum

bound to silicates. The method, especially using pneumatic nebulization,
may not achieve quantitative measurement of typical concentrations in
some foods for some elements. Using ultrasonic nebulization will
improve analytical limits for most elements. The following elements
appear prone to laboratory environmental contamination and therefore
require extensive assessment of contamination control: aluminum,
chromium, and lead.
Caution 1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
3) Reagents should be regarded as potential health hazards and exposure
to these materials should be minimized. Follow universal precautions.
Wear gloves, a lab coat, and safety glasses while handling reagent.
4) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
5) Microwave digestion systems are dangerous. Vessels contain
concentrated nitric acid at high temperatures and pressures. Analyst
must be familiar with manufacturer’s recommended safety
precautions. Never remove hot vessels from microwave; wait until
they are near room temperature. Keep microwave door closed while
vessels are hot. The door is the primary safety device if a vessel vents.
Principle An analytical portion (0.4 to 5 g dependent on food composition) is
decomposed with nitric acid and hydrogen peroxide in a high-pressure
Teflon® lined digestion vessel using microwave heating and a feedback
program to control temperature and pressure. A 50 mL analytical solution
is prepared from the digest. Analytical solutions are nebulized and aerosol
is transported to plasma where desolvation and excitation occur. Either
pneumatic or ultrasonic nebulization sample introduction is used.

4
 Characteristic atomic emission spectra are produced by radio frequency
inductively coupled plasma. Spectra are dispersed by a grating
spectrometer, and line intensities are measured with a light sensitive
detector such as a photomultiplier tube or charge transfer device.
Photocurrents are processed by a computer system. A background
correction technique is required to compensate for variable background
emission contribution to analyte signal and should be applied except in
cases of line broadening.
Apparatus/Instruments 1) Inductively coupled plasma atomic emission spectrometer (ICP-
AES)—Simultaneous or sequential ICP-AES with associated
glassware, which uses a mass flow controller to regulate argon
nebulizer flow rate supplied by a Dewar of liquid argon or tank of
gaseous argon. A variable speed peristaltic pump to deliver all
solutions to nebulizer. Pneumatic nebulizer which can aspirate high
dissolved solids (e.g., V-groove, cross flow, etc.) or an ultrasonic
nebulizer.
2) Microwave decomposition system—requires temperature control to
200 °C, pressure control to at least 600 psi, power range of 0-100% in
1% increments, minimum 1000 watts for 12 position carousel,
feedback control of temperature and pressure and multistep
programming with ramp to temperature capability. Digestion vessels
must be quartz or Teflon lined. System must be able to reach at least
200 °C and at least 600 psi. Vessels designed to vent and reseal can be
used provided they vent at pressures >300 psi.
Materials and Reagents Reagents may contain elemental impurities that can affect the quality of
analytical results. Use of high purity or trace element (i.e., metals) grade
reagents is usually required.
1) Reagent water—Water that meets specifications for ASTM Type I
water
2) High purity nitric acid—concentrated (sp gr 1.41), trace element grade
or double distilled.
3) Nitric acid—Concentrated (sp gr 1.41), ACS reagent grade.
4) Nitric acid 1% (v/v)—Dilute 10 mL high purity nitric acid to 1000 mL
with reagent water.
5) (5) Nitric acid 10% (v/v)—Dilute 100 mL high purity nitric acid to
1000 mL with reagent water.
6) (6) Hydrogen peroxide—30% H2O2 solution. High purity or trace
metals grade.
Preparation of Reagents 1) Stock standard solutions—Commercially prepared single element

5
 solutions prepared specifically for spectrometric analysis (usually
1000 or 10,000 mg/L). Stock standard solutions may also be prepared
in the laboratory from high purity (≥99.99%) metals or salts.
Alternatively, commercial multi-element solutions prepared
specifically for spectrometric analysis can be used. These multi-
element solutions will be much lower in concentration (typically 10-
500 mg/L) than single element solutions to avoid compatibility
problems.
2) Intermediate standard solution(s)—Prepared to contain appropriate
concentration(s) of analytes for preparation of standard solutions. Pipet
an appropriate volume of stock standard solution(s) into an acid rinsed
volumetric flask and dilute to volume with 10% nitric acid. Store
prepared intermediate standard solutions in plastic bottles.
Alternatively, commercial multi-element solutions prepared
specifically for spectrometric analysis can be used.
3) Standard solutions—prepare at least 3 standard solutions by
combining appropriate volumes of stock standard solutions or
intermediate standard solutions in volumetric flasks. Analyte
concentration range should cover the LDR or a portion thereof. Dilute
to volume with 10% nitric acid. Many of the elements (cadmium,
cobalt, molybdenum, etc.) have LDRs that far exceed the values
expected in food analytical solutions. In addition, line-rich elements
like iron may cause spectral interference on other emission lines if
high concentrations are used to standardize the instrument. Therefore,
the analyst may choose to work within part of the LDR. A
recommended maximum concentration of an element in a standard
solution is 10 mg/L. Exceptions would be elements usually present at
high concentrations for example, calcium, sodium, potassium,
magnesium and phosphorus. For convenience, each standard solution
should contain all the analytes to be determined.
Chemical compatibility (i.e., of analytes, acids, etc.) must be
considered to avoid the formation of analyte precipitates when mixing
single element stock solutions to prepare standard solutions. High
quality custom-made multi-element solutions are commercially
available and are recommended. Transfer prepared standard solutions
to acid cleaned plastic bottles (Teflon FEP is preferred) for storage.
4) Standard blank—10% nitric acid. Prepare sufficient amount for use
in standardization, determination of IDLs, and for nebulizer rinse
between each measurement.

6
 5) Independent check solution (ICS)—Dilute appropriate volumes of
analyte stock solutions or intermediate standard solutions obtained
from a different source than used to prepare standard solutions with
10% nitric acid so analyte concentration will be several times the
ASQL or in the range of 0.5 to 10 mg/L for most elements.
6) Check solution—Use mid-concentration multi-analyte standard
solution for the check solution.
7) Spike solution—Prepared such that, when 1 mL is diluted to
analytical solution volume (initial analytical solution volume usually
50 mL), analyte concentration is approximately at the middle of the
LDR or appropriate for the expected sample analyte concentration. A
fortification solution should not be prepared that would result in an
analyte concentration in the analytical solution that is less than 10
times the ASQL. In addition, the fortification solution should not
increase any analyte’s concentration by more than 40 mg/L relative to
the analytical solution because of potential problems caused by high
analyte levels (nebulizer transport effects and spectral interference,
etc.) and the challenge of minimizing the spike solution volume. Pipet
an appropriate volume of stock standard solution(s) or intermediate
standard solution(s) into an acid rinsed volumetric flask and dilute to
volume with 10% nitric acid.
Sample Preparation 1) Weigh analytical portion into clean vessel liner and determine mass of
analytical portion. Generally, for samples of unknown composition,
weight the equivalent of about 0.5 dry material to an accuracy of 0.001
g. Less than the maximum mass should be used for samples high in
salt content. A maximum analytical portion of 5 g should not be
exceeded even if calculations based on the food’s energy indicate that
a larger portion could be taken. Use 1 g reagent water for method
blanks (MBKs). For dry samples and dry CRM materials adding 1 g of
reagent water can help control exothermic reactions during the
digestion.
2) Pipette 8.0 mL or weigh 11.3 g of high purity nitric acid (sp gr 1.41
g/mL) into vessel liner, washing down any material on walls.
Weighing acid using a top loading balance and Teflon FEP wash bottle
is suggested. Use double distilled grade for lowest method blank
values. Acid should be added drop wise for the first few mL until it
can be established that the sample will not react violently. Some foods,
especially those high in sugar, will react with nitric acid within several
minutes. If foaming or reaction with the acid is observed, let the

7
 vessels sit uncovered in a class 100 clean hood for 20 minutes or until
reaction subsides. If a clean hood is unavailable, place caps on vessels
without pressing down fully or, if so equipped, cap vessels but loosen
the pressure relief nut (with the safety membrane) to allow pressure to
escape. If, however, it appears that excessive foaming would result in
the sample-acid mixture expanding out of the vessel then cap the
vessel and tighten to appropriate torque to prevent loss of sample or
acid.
3) Add 1 mL high purity 30% H2O2. Seal vessels, apply correct torque to
cap (tighten pressure relief nuts if equipped) and run the digestion
program as given in table.

Digestion program with Ramp to Temperature feature

and pressure control
Maximum Power (Watts) 1200
Control Pressure (psi) 800
Ramp Time (min) 25
Hold Time (min) 15
Control Temperature (°C) 200
Power is applied for the Ramp Time minutes or until Control
Pressure or Control Temperature is met. If Control Pressure or
Control Temperature are met before end of Ramp Time then
program proceeds to Hold Time.

4) After vessels have cooled to less than 50° C remove them to an

exhausting clean hood and vent excess pressure slowly. Quantitatively
transfer and dilute digestion solution with reagent water to 25 mL.
This analytical solution should be transferred to a plastic bottle or a
capped polypropylene centrifuge tube for storage.
Method of analysis 1) Instrument Setup -Setup inductively coupled plasma optical emission
spectrometer according to the manufacturer’s recommendations and
with the following attributes:
• Set rinse time to at least 60 sec.
• Program instrument method for the analytes of interest. Include
the following elements even if they are not analytes of interest to
allow for interference correction: Al,Ca, Fe, Cr, Cu, Mn, Ti, and
V.
• Suggested emission line wavelengths are listed in below table.
Other wavelengths may be used but they may not achieve the
same sensitivities.

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 Element Wavelength (nm)
Aluminum (Al) 308.22
Arsenic (As) 189.01
Barium (Ba) 493.41
Boron (B) 249.68
Cadmium (Cd) 226.50
Calcium(Ca) 317.93
Chromium (Cr) 267.72
Cobalt (Co) 228.62
Copper (Cu) 324.75
Iron (Fe) 259.94
Lead (Pb) 220.35
Magnesium (Mg) 383.83
Manganese (Mn) 257.61
Molybdenum (Mo) 202.03
Nickel 231.60
Phosphorus (P) 178.29
Potassium (K) 766.49
Sodium (Na) 589.59
Strontium (Sr) 407.77
Thallium (Tl) 190.86
Vanadium (V) 292.40
Zinc (Zn) 213.86

• Use background correction.

• Configure instrument for 3 integrations of emission. Use
integration time appropriate for the particular instrument and
emission line. Allow at least 10 sec after the solution reaches the
plasma before starting integration. Report each emission reading
and the mean and RSD.
• Program instrument to use a linear, least squares calculated
intercept, curve fit algorithm for converting emission values to
mg/L concentration units. Do not subtract standard blank response
from standard solution response. Use the mean of the emission
integrations to calculate concentration of analyte.

2) Determination of Analyte Concentration Using Standard Curve

i. Standardize the instrument using the standard blank and at least 3
standard solution concentration levels. Allow at least 10 sec after
the standard solution reaches the plasma before starting

9
 integration. Flush system with standard blank for at least 60 sec
between each standard solution.

ii. Check Standardization Performance

• Correlation coefficient (r) of linear regression (emission intensity

verses concentration) is ≥0.998.
• ICS recovery within 100 ± 5% (initial calibration verification).
• Standard blank <ASDL.

iii. Analyze analytical solutions and quality control solutions.

Interpolate analyte concentration from standard curve. Rinse
sample introduction system by aspirating standard blank for a
minimum of 60 sec between all analyses (or longer if necessary).
Rinse time is appropriate if results of a standard blank are <ASDL
when analyzed immediately after a high standard.

iv. Check Instrument Measurement Performance

• RSD of replicate integrations ≤7% for all solutions when
instrument response ≥ASQL.
• Check solution analyzed at a frequency of 10% and at the end of
the analytical run has a recovery of 100 ± 10% (continuing
calibration verification).
• Standard blank analyzed at a frequency of 10% and at the end of
the analytical run <ASDL (continuing calibration blank).
• Measurements are below highest standard solution. Dilute
analytical solution with standard blank if necessary to comply with
criteria.
• Wavelength scan indicates absence of spectral interference that is
not corrected for by background correction or inter-element
correction factors.

v. Inter-element Correction Factors

• If analytical solution has or is expected to have Al, Ca, Fe, Cr,
Cu, Mn, Ti or V at concentrations >20 mg/L then inter-element
correction factors must be determined as outlined in
manufacturer's Instructions. Program instrument to use these
factors.
• Analyze the solution(s) used to determine the inter-element
correction factors as a sample to demonstrate proper correction for
interference.

Calculation with units of Calculate the concentration (mass fraction) of the analyte in the analytical
expression portion according to the formula

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 Concentration (mg/kg)=[(S×DF)−MBKL]×V
m×MCF
where
S = concentration of analyte in analytical solution (or diluted
analytical solution) (mg/L)
MBKL = laboratory MBK (mg/L)
V = volume (L) of analytical solution (usually 0.050 L)
m = mass of analytical portion (kg)
DF = dilution factor (1 if analytical solution not diluted)
MCF = mass correction factor (1 if no water or other solvent was
added to aid homogenization)
Round calculated concentration to at most 3 significant figures.
Concentration may be converted to other convenient units (e.g., μg/kg,
ng/kg).
Inference
(Qualitative Analysis)
Reference U.S. Food and Drug Administration-(4.8) High Pressure Liquid
Chromatographic-Inductively Coupled Plasma-Mass Spectrometric
Determination of Methylmercury and Total Mercury in Seafood (version
1.1) (August 1010)
Approved by Scientific Panel on Methods of Sampling and Analysis

11
 Method for Determination of Calcium, Copper, Iron, Magnesium,
Manganese, Potassium, Phosphorus, Sodium, and Zinc in Fortified
Food Products by Inductively Coupled Plasma-Optical Emission
Spectrometry (ICP-OES)
Method No. FSSAI 09.002:2024 Revision No. & Date 0.0
Scope Applicable to analysis of calcium, copper, iron, potassium, magnesium,
manganese, phosphorus, sodium, and zinc in fortified food products.
Limit of quantitation (LOQ; mg/kg):Ca (150); Cu (2); Fe (10); K (200);
Mg (50); Mn (0.05); Na (100); P (100); Zn (5).
Caution 1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
3) Reagents should be regarded as potential health hazards and exposure
to these materials should be minimized. Follow universal precautions.
Wear gloves, a lab coat, and safety glasses while handling reagent.
4) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
5) Application of microwave digestion systems involves hot pressurized
acid solutions and concentrated acids. Follow manufacturer’s
directions for safety risk and safety environment of microwave
systems. Never remove hot vessels from microwave; wait until they
are near room temperature. Keep microwave door closed while vessels
are hot. The door is the primary safety device if a vessel vents.
Principle The principle involves the removal of organic matter of the sample
through acid digestion to ensure the trace elements are in free form for
their measurement by ICP-OES. Test portion is heated at 200°C either
with nitric acid in a closed-vessel microwave digestion system (MDC) or
with a combination of hydrogen peroxide, nitric acid, and hydrochloric
acid in an open-vessel microwave digestion system (MDO).
Apparatus/Instruments 1) Microwave- Commercial MDC or MDO designed for laboratory use at
200 ± 20°C, up to 600 psi, and controlled temperature or pressure
ramping capability. It is recommended that vessel design be selected
that will withstand the maximum possible pressure (600 psi) since
organic residues of rich-fat or rich-carbohydrate samples, if not given
sufficient time to predigest, will generate significant pressure during
digestion.
2) ICP-OES spectrometer—Instrument with axial, radial, or dual view

12
 grating configurations and auto sampler, capable of determining
multiple wavelengths for each element of interest with the required
sensitivity. A 3-channel peristaltic pump with or without appropriate
in-line addition system (e.g., T connector) are linked between the
peristaltic pump and nebulizer to avoid having to manually add
ionization buffer and internal standards to each sample solution. A
thermostated cyclonic spray chamber equipped with a micro-
concentric nebulizer or other components designed to optimize aerosol
and maximize precision was used.
Ionization buffer (cesium chloride) is combined with the internal
standard solution to compensate EIEs effects (e.g., K, Na, and Ca) in
the plasma since certain food materials can contain substantial
concentrations of these elements. This provides a significant source of
electrons in the plasma. The presence of ionization buffer in all
samples and standards will minimize the effects of varying
concentrations of EIEs in the sample. The solution presented to the
nebulizer contains a maximum of 5000 mg/kg cesium for high- salted
food raw materials (e.g., culinary products or tastemakers) and a
minimum of 500 mg/kg cesium (for main food samples); 20 mg/kg
indium and 5 mg/kg strontium, yttrium, and chromium; less than half
of each element concentration of the higher working standard Std6 and
less than 0.5 g/kg total dissolved minerals.
3) ICP wavelengths- A number of recommended and alternative
wavelengths may be used for the nine elements to be determined and
internal standards. As a minimum, select one recommended and one
alternative wavelength for each element corrected by one
recommended wavelength for appropriate internal standard. All
responses for both recommended and alternative wavelengths for each
element are corrected using only one internal standard line. The
following is a list of wavelengths for each element (and its appropriate
internal standard) in priority order that have been found acceptable for
main foodstuffs:

The following is a list of wavelengths for each element (and its

appropriate internal standard) in priority order that have been found
acceptable for main foodstuffs: Wavelength (nm): Ca: 317.933 (In:
303.936); Cu: 324.754 (In: 303.936); Fe: 259.94 (Sr: 338.071); K:
766.491 (Sr: 460.733); Mg: 285.213 (In: 303.936); Mn: 257.610 (Sr:
338.071); Na: 589.592 (Sr: 460.733); P: 213.618 (In: 303.936); Zn:
213.857 (Sr: 338.071).
Other wavelengths that are acceptable for both elements and internal
standards could be used as confirmatory analytical lines or alternative
wavelengths as certain recommended lines may not be available on
some ICP-OES systems: Wavelength (nm): Ca: 317.933 (Y: 371.028);
Cu: 324.754 (Y: 371.028), 327.395 (In: 303.936 or Y: 371.028); Fe:

13
 259.94 (Y: 371.028 or Cr: 283.563); Mg: 285.213 (Y: 371.028),
279.028 (In: 303.936); Mn: 257.610 (Sr: 460.733 or Y: 371.028); P:
178.222 (Sr: 460.733 or Y: 371.028); Zn: 213.857 (Sr: 460.733 or Y:
371.028).
Materials and Reagents 1) High-grade water, (18 MΩ).—For slurry preparation and/or dilution.
2) Nitric acid (HNO3), 65% (w/v).—Trace metal grade throughout.
3) Hydrochloric acid (HCl), 37% (w/v).—Trace metal grade throughout.
4) Hydrogen peroxide (H2O2), 97% (w/v).—Trace metal grade
throughout.
Preparation of Reagents (a) Ionization buffer/internal standard solution—Weigh 1.27 g
cesium chloride into a 1000 mL acid-washed volumetric flask [Note:
This cesium 0.1% (w/v) solution was tested as the minimal
recommended concentration required for element analysis in most
food matrixes. Cs solution 1% (w/v) is recommended if an element is
present at low concentration in high-salted food raw materials, e.g.,
culinary products or tastemakers, or if it is analyzed as an impurity in
food-grade salts.] Add 40 mL indium 1000 mg/kg and 10 mL each of
strontium, yttrium, and chromium 1000 mg/kg stock standard
solutions, as internal standards. Add 10 mL HNO3. Dilute to volume
with H2O, mix, and transfer to an acid-washed polyethylene bottle.
(Note: Reagent concentrations assume the use of same pump tubing
internal diameter for both internal standard/ionization buffer and
sample pump tubes using automatic addition.)
(b) Stock standard solution—Working standards can be prepared from
ICP-grade individual element 1000 mg/kg (e.g., for copper, iron,
manganese, and zinc) and 10 000 mg/kg (e.g., for calcium,
magnesium, phosphorus, potassium, and sodium) commercial stock
standard solutions. However, it is also acceptable to use
commercially prepared, custom blended stock standard mixtures
containing all of the nine elements at appropriate concentrations.
(c) Intermediate stock solution—Suggested composition of the
intermediate stock standard solution:
Table 1 (Preparation of intermediate solutions from stock solution)
Stock Intermedia Volume of
S.No. Metal solution te stock stock solution
conc. solution required for
(mg/kg) conc. 500 ml
(mg/kg)
1 Calcium 10000 1500 75
2 Magnesium 10000 500 25

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 3 Phosphorus 10000 1000 50
4 Potassium 10000 2000 100
5 Sodium 10000 1000 50
6 Copper 1000 10 5
7 Iron 1000 50 25
8 Manganese 1000 0.25 0.125
9 Zinc 1000 20 10

(d) Working standard solutions—Standards prepared from intermediate

stock standard solution are designed to have the same acid
concentration as digested test solutions (i.e., 10%, v/v, HNO3) for
MDC or 15% (v/v) for MDO using combined acids (HNO3, H2O2,
and HCl).
(1) Std6—Pipet 15.0 mL intermediate stock standard solution into a
100 mL acid-washed volumetric flask. Add 10 mL HNO3 (MDC) or
15 mL combined acids (MDO), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle.
(2) Std5—Pipet 10 mL intermediate stock standard solution into a
100 mL acid-washed volumetric flask. Add 10 mL HNO3 (MDC) or
15 mL combined acids (MDO), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle.
(3) Std4—Pipet 5.0 mL intermediate stock standard solution into a
100 mL acid-washed volumetric flask. Add 10 mL HNO3 (MDC) or
15 mL combined acids (MDO), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle.
(4) Std3—Pipet 2.0 mL intermediate stock standard solution into a
100 mL acid-washed volumetric flask. Add 10 mL HNO3 (MDC) or
15 mL combined acids (MDO), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle.
(5) Std2—Pipet 1.0 mL intermediate stock standard solution into a
100 mL acid-washed volumetric flask. Add 10 mL HNO3 (MDC) or
15 mL combined acids (MDO), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle.
(6) Std1—Pipet 0.5 mL intermediate stock standard solution into a
100 mL acid-washed volumetric flask. Add 10 mL HNO3 (MDC) or
15 mL combined acids (MDO), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle.
(7) Blank—Add 10 mL HNO3 (MDC) or 15 mL combined acids
(MDO) into a 100 mL acid-washed volumetric flask, dilute to
volume with H2O, mix, and transfer to acid-washed polyethylene

15
 bottle. All calibration solutions when made are stable for 1 week in
glass volumetric flasks.
(e) Sampler wash solution- 10% HNO3 (v/v).—Dilute 100 mL trace
metal-grade HNO3 to 1000 mL with H2O.
Table-2 (Suggested concentration of the six standard solutions,
mg/kg)
Element Blank Std 1 Std 2 Std 3 Std 4 Std 5 Std 6
Calcium 0 7.5 15 30 75 150 225
Magnesiu 0 2.5 0.5 10 25 50 75
m
Phosphoru 0 5 10 20 50 100 150
s
Potassium 0 10 20 40 100 200 300
Sodium 0 5 10 20 50 100 150
Copper 0 0.05 0.1 0.2 0.5 1.0 1.5
Iron 0 0.25 0.5 1.0 2.5 5 7.5
Manganes 0 0.001 0.002 0.005 0.012 0.025 0.037
e 25 5 5 5
Zinc 0 0.1 0.2 0.4 1.0 2.0 3.0

Sample Preparation (a)Sample preparation—

(1) Test sample preparation—Homogenize a representative sample by
grinding as finely as possible and/or by preparing a slurry with H2O.
For example: Infant cereals and fortified milk powders, preheat water
at 50°C. Prepare the slurry by weighing 10.0 ± 0.1 g test sample and
place into a 100 mL Erlenmeyer flask; add 90.0 ± 0.1 g H2O. Mix
well with stopper.

(2) Test portion preparation—Accurately weigh 0.50 ± 0.01 g test

portion or sample mass on a dry weight basis in the prepared slurry
to MDC vessel (1.00 ± 0.01 g into a 100 mL volumetric flask for
MDO). [Note: An optimal analytical test portion mass of 0.5 g (1.0 g
for MDO) is based on an empirical maximum energy release by the
food of 3 kcal and 90–110% recovery.]
Line the MDC vessel walls or Pasteur pipet with weighing paper
during sample transfer to keep sample from adhering to sides of
vessel or use a Pasteur pipet to transfer liquid samples. (Weigh fluid
samples or test portion from slurry test sample directly after mixing.)
(Note: Remove weighing paper from sample prior to next step.)
Carefully add 5.0 ± 0.1 mL HNO3 into MDC/MDO vessel (and then
5 mL H2O2 only into MDO vessel).Loosely cap MDC vessel

16
 without sealing. Predigest for at least 10 min at room temperature or
until vigorous foaming subsides.
Close MDC vessels and distribute onto microwave carousel to ensure
uniform microwave power application on all samples.
(3) Food-grade salt sample preparation—Weigh 0.20 ± 0.01 g food-
grade salt (a minimum dilution factor of 500 is recommended) into a
100 mL volumetric flask. Add deionized water and 10 mL HNO3.
Dissolve salt and dilute to volume with deionized water.
(b) Test portion digestion—
(1) Sample digestion—With power setting appropriate to MDC
(maximum power of 1600 W) and MDO models (maximum power
of 600 W), and number of vessels used, heat MDO vessels at 200
± 20°C for 20 min or ramp MDC temperature from ambient to 200
± 20°C in 15 min and hold at 200°C for 25 min.
(Note: Yellow vapors will be emitted during the hydrolysis in MDO
vessels.)
Carefully remove the MDO vessels. Allow the vessels to cool down
to room temperature.
Add 5 ml HCl 35% (w/v) into MDO vessels and heat MDO vessels
at 200 ± 20°C for 20 min.
Cool vessels to room temperature before venting (MDC vessels).
Transfer the MDC digests to 50 mL (100 mL for MDO) volumetric
flasks. Dilute to volume with H2O and mix. (Note: A digestion is
judged complete when clear to yellow analytical solutions are
produced.)
Filter the digested solution using an ashless filter paper for turbid
samples containing fat. Discard the first 20 mL filtrate and collect the
remaining filtrate for analysis.
(Note: Membrane disc filters (0.45 μm) are not recommended as they are
generally not metal-free.)
Transfer to polyethylene containers within 2 h.
Dilute the samples that are found to be above the standard curve
range or have total content of minerals higher than 1000 mg/L with
H2O.

Method of analysis 1) Make a calibration curve using either weighted linear or quadratic
regression with correlation coefficients of at least 0.9999 from seven
standards prepared from intermediate standard solution, including a
blank (Std 0) and six suggested concentrations of the standard
solution (Std1–Std6) shown in Table-2and expressed in mg/kg.
2) Analyze test solutions using an ICP-OES instrument calibrated with
the working standard solutions.
3) Insert a working standard or other suitable quality control solution

17
 every 10 test portions to monitor for instrument drift. The inclusion
of a digestion blank, a sample duplicate, and known reference
materials is highly encouraged.

Calculation with units of The concentration (C) of each element, in mg/kg, is calculated as follows:
expression axVxF
C=
m
where
C = concentration in the test portion sample (mg/kg);
a = concentration (mg/L) of the element in the digest solution as
obtained from instrument;
V = volume (mL) of the test solution after being made up (i.e., 50
mL for MDC and 100 mL for MDO);
F = dilution factor of the test solution;
m = weight of the test portion (g).
Inference
(Qualitative Analysis)
Reference AOAC Official Method 2011.14
Approved by Scientific Panel on Methods of Sampling and Analysis

18
 Method for the analysis of trace elements in Water by Inductively
Coupled Plasma Mass Spectrometry (ICP-MS)

Method No. FSSAI 09.003:2024 Revision No. & Date 0.0

Scope This method specifies the determination of the elements aluminium,
antimony, arsenic, barium, beryllium, bismuth, boron, cadmium, cesium,
calcium, cerium, chromium, cobalt, copper, dysprosium, erbium,
europium, gadolinium, gallium, germanium, gold, hafnium, holmium,
indium, iridium, lanthanum, lead, lithium, lutetium, magnesium,
manganese, molybdenum, neodymium, nickel, palladium, phosphorus,
platinum, potassium, praseodymium, rubidium, rhenium, rhodium,
ruthenium, samarium, scandium, selenium, silver, sodium, strontium,
terbium, tellurium, thorium, thallium, thulium, tin, tungsten, uranium,
vanadium, yttrium,
ytterbium, zinc, and zirconium in water [for example drinking water,
surface water, groundwater, wastewater and elutes.

The working range depends on the matrix and the interferences

encountered. In drinking water and relatively unpolluted waters, the limit
of application is between 0.1μg/l and 1.0 μg/l for most elements.

The detection limits of most elements are affected by blank contamination

and depend predominantly on the laboratory air-handling facilities
available.
Caution
1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
3) Reagents should be regarded as potential health hazards and exposure
to these materials should be minimized. Follow universal precautions.
Wear gloves, a lab coat, and safety glasses while handling reagent.
4) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
Principle Multi-element determination of 62 elements by inductively coupled
plasma mass spectrometry (ICP-MS) consists of the following steps:

-introduction of a measuring solution into a radiofrequency plasma (for

example by pneumatic nebulization) where energy transfer processes
from the plasma cause dissolution, atomization and ionization of

19
 elements;

-extraction of the ions from plasma through a differentially pumped

vacuum interface with integrated ion optics and separation on the basis
of their mass-to-charge ratio by a mass spectrometer (for instance a
quadrupole MS);

-transmission of the ions through the mass separation unit (for instance
a quadrupole) and detection, usually by a continuous dynode electron
multiplier assembly, and ion information processing by a data handling
system;

-quantitative determination after calibration with suitable calibration

solutions.

The relationship between signal intensity and mass concentration is

usually a linear one over at least five orders of magnitude.
Apparatus/Instruments The stability of samples, and measuring and calibration solutions depends
to a high degree on the container material. The material shall be checked
according to the specific purpose. For the determination of elements in a
very low concentration range, glass or polyvinyl chloride (PVC) should
not be used. Instead, it is recommended to use perfluoroalkoxy (PFA),
hexafluoroethene propene (FEP) or quartz containers, cleaned with hot,
concentrated nitric acid in a closed system. For the determination of
elements in a higher concentration range, high density polyethene
(HDPE) or polytetrafluoroethene (PTFE) containers are also allowed for
the collection of samples.

Immediately before use, all glassware should be washed thoroughly with

warm diluted nitric acid [for example w(HNO3) = 10 %], and then rinsed
several times with water.

The use of piston pipettes is permitted and also enables the preparation of
lower volumes of calibration solutions. The application of dilutors is also
allowed. Every batch of pipette tips and disposable plastics vessels shall
be tested for impurities.

(1) Mass spectrometer.

A mass spectrometer with inductively coupled plasma (ICP) suitable
for multi-element and isotope analysis isrequired. The spectrometer
should be capable of scanning a mass range from 5 m/z (AMU) to 240
m/z (AMU)
with a resolution of at least 1 mr /z peak width at 5 % of peak height
(mr = relative mass of an atom species;
z = charge number).

20
 The instrument may be fitted with a conventional or extended
dynamic range detection system.

(2) Mass-flow controller.

A mass-flow controller on the nebulizer gas supply is required. Mass-
flow controllers for the plasma gas and the auxiliary gas are also
useful. A water cooled spray chamber may be of benefit in reducing
some types of
interferences (for example from polyatomic oxide species).

NOTE The plasma is very sensitive to variations in the gas flow rate.

(3) Nebulizer with variable speed peristaltic pump, for which

information on different types of nebulizers
is given in ISO 17294-1:—, 5.1.2.

(4) Argon gas supply, of high purity grade, for instance 99,99 %.

(5) Glassware, consisting of the following:

-volumetric flasks, for example 50 ml, 100 ml, 500 ml and 1 000 ml;
-conical (Erlenmeyer) flasks, for example 100 ml;
-pipettes, for example 1 ml, 2,5 ml, 10 ml, 20 ml and 25 ml.

(6) Storage bottles, for the stock, standard, calibration and sample
solutions.
For the determination of elements in a normal concentration range,
high density polyethene (HDPE) or polytetrafluoroethene (PTFE)
bottles are sufficient for the storage of samples. For the determination
of elements in an ultratrace level bottles made from perfluoroalkoxy
(PFA) or hexafluoroethene propene (FEP) should be preferred. In any
case the user has to check the suitability of the chosen containers.
Materials and Reagents For the determination of elements at trace and ultratrace level, the
reagents shall be of adequate purity. The concentration of the analyte or
interfering substances in the reagents and the water should be negligible
compared to the lowest concentration to be determined.

For preservation and digestion, nitric acid should be used to minimize

interferences by polyatoms.

1) Water, Grade 1,for all sample preparation and dilutions.

2) Nitric acid, sp.g.(HNO3) = 1,4 g/ml.
3) Hydrochloric acid, sp.g. (HCl) = 1,16 g/ml.
4) Hydrochloric acid, dilute(HCl) = 0,2 mol/l.
5) Sulfuric acid, sp.g.(H2SO4) = 1,84 g/ml.
6) Hydrogen peroxide, w(H2O2) = 30 %.

21
 7) Element stock solutions,sp.g.= 1 000 mg/l each of Ag, Al, As,
Au, B, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr, Cs,Cu, Dy, Er, Eu, Ga, Gd,
Ge, Hf, Ho, In, Ir, K, La, Li, Lu, Mg, Mn, Mo, Na, Nd, Ni, P, Pb,
Pd, Pr, Pt, Rb, Re, Rh, Ru, Sb, Sc, Se, Sm, Sn, Sr, Tb, Te, Th, Tl,
Tm, U, V, W, Y, Yb, Zn, Zr.
Both single-element stock solutions and multi-element stock solutions
with adequate specification stating the acid used and the preparation
technique are commercially available. Element stock solutions with
different concentrations of the analytes (for example 1000 mg/l) are also
allowed.

Anion stock solutions, = 1 000 mg/l each of Cl, PO4-3, SO4-2.

Prepare these solutions from the respective acids. The solutions are also
commercially available. Anion stock solutions with different
concentrations of the analytes (for example 100 mg/l) are also allowed.

Preparation of Reagents 1) Multi-element calibration solutions

Depending on the scope, different multi-element standard solutions may
be necessary. In general, when combining multi-element standard
solutions, their chemical compatibility and the possible hydrolysis of the
components shall be regarded. Care shall be taken to prevent chemical
reactions (for example precipitation).

The multi-element standard solutions are considered to be stable for

several months, if stored in the dark.

This does not apply to multi-element standard solutions that are prone to
hydrolysis, in particular solutions of Bi, Mb, Mo, Sn, Sb, Te, W, Hf and
Zr.
(a) Multi-element standard solution A, consisting of the following:
ρ(As, Se) = 20 mg/l
ρ (Ag, Al, B, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr, Cs, Cu, La, Li, Mg, Mn,
Ni, Pb, Rb, Sr, Th, Tl, U, V, Zn)= 10 mg/l

Pipette 20 ml of each element stock solution (As, Se) and 10 ml of

each element stock solution (Ag, Al, B,Ba, Be, Bi, Cd, Ce, Co, Cr, Cs,
Cu, La, Li, Mn, Ni, Pb, Rb, Sr, Th, Tl, U, V, Zn) into a 1 000 ml
volumetricflask.

Add 10 ml of nitric acid.

Bring to volume with water and transfer to a suitable storage bottle.

Multi-element standard solutions with more elements may be used

provided it is verified that these solutions are stable and no chemical
reactions occur. This shall be checked again a few days after the first

22
 use
(sometimes precipitation can occur after preparation)

(b) Multi-element standard solution B, consisting of the following:

ρ (Au, Mo, Sb, Sn, W, Zr) = 5 mg/l.

Pipette 2,5 ml of each element stock solution (Au, Mo, Sb, Sn, W, Zr)
into a 500 ml volumetric flask.
Add 40 ml of conc. hydrochloric acid.
Bring to volume with water and transfer to a suitable storage bottle.

(c) Reference-element solution (internal standard solution).

The choice of elements for the reference-element solution depends on

the analytical problem. Solutions of these elements should cover the
mass range of interest. The concentrations of these elements in the
sample
should be negligibly low.
The elements In, Lu, Re, Rh and Y have been found suitable for this
purpose.

For example, the following reference-element solutions may be used:

ρ(Y, Re) = 5 mg/l
Pipette 5 ml of each element stock solution (Y, Re) into a 1000 ml
volumetric flask.
Add 10 ml of nitric acid.
Bring to volume with water and transfer to a suitable storage bottle.

2) Multi-element calibration solutions.

Choose the mass concentrations of the calibration solutions to allow for
a sufficient precision and reproducibility and ensure that the working
range is covered.

The stability of the calibration solutions should be checked regularly.

Due to their rather low respective mass concentrations, they should be
replaced by freshly prepared solutions at least every month or more
frequently for elements which are prone to hydrolysis. In special cases,
daily preparation is necessary. The user has todetermine the maximum
stability period of the calibration solutions.

Transfer the calibration solution(s) A and B to suitable storage bottles.

If the determination is carried out after previous digestion the matrix of
the multi-element calibrationsolution(s) A and B shall be adjusted to
that of the digests.
The working range in general may cover the range of 0.1 μg/l to 50

23
 μg/l or a part of this.
(a) Multi-element calibration solution(s) A.
Prepare the calibration solution(s) A that cover the required working
range by diluting the multi-element standard solution A.

Add 10 ml of nitric acid per litre and bring up to volume with water.
If necessary, add reference-element solutionto a concentration of for
example 50 μg/l of the reference elements before bringing up to
volume.

(b) Multi-element calibration solution(s) B.

Prepare the calibration solution(s) B that cover the required working
range by diluting the multi-elementstandard solution B
Add 5 ml of hydrochloric acid per litre and bring up to volume with
water.
If necessary add reference-element solution to a concentration of, for
example, 50 μg/l of the reference-elements before bringing up to
volume.
3) Blank calibration solutions.
High demands shall be set concerning the purity. The user should
ensure that the background levels of the analytes are not significant to
the results of the analysis.

(a) Blank calibration solution A.

Pipette 0,5 ml of nitric acid to a 100 ml volumetric flask made for
example from perfluoroalkoxy (PFA) or hexafluoroethene propene
(FEP) and bring to volume with water. If necessary, add reference-
elementsolution to a concentration of, for example, 50 μg/l of the
reference-elements before bringing up to volume.
If the determination is carried out after previous digestion the matrix
of the blank calibration solution A shall be adjusted to that of the
digests.

(b) Blank calibration solution B.

Pipette 1,0 ml of conc. hydrochloric acid to a 100 ml volumetric flask
made for example from perfluoroalkoxy (PFA) or hexafluoroethene
propene (FEP) and bring to volume with water. If necessary add
reference elementsolution to a concentration of for example 50 μg/l of
the reference-elements before bringing up to volume.

If the determination is carried out after previous digestion the matrix

of the blank calibration solution Bshall be adjusted to that of the
digests.

4) Optimization solution.

24
 The optimization solution serves for mass calibration and for
optimization of the apparatus conditions, for example adjustment of
maximal sensitivity with respect to minimal oxide formation rate and
minimal formation of doubly charged ions.
It should contain elements covering the entire mass range, as well as
elements prone to a high oxide formation rate or to the formation of
doubly charged ions. For example, an optimization solution containing
Mg, Cu, Rh, In, Ba, La, Ce, U and Pb is suitable. Li, Be and Bi are less
suitable because they tend to cause memory effects.

The mass concentrations of the elements used for optimization should

be chosen to allow count rates of more than 10 000 counts/s.

5) Matrix solution.
The matrix solutions serve to determine the correction factors for the
corresponding equations. High demandsare made concerning the purity
of the basic reagents due to the high mass concentrations. The user
shouldensure that the background levels of the analytes in the matrix
solution are not significant to the results of theanalysis. The
composition may be as follows:
ρ(Ca) = 200 mg/l;
ρ(Cl-) = 300 mg/l;
ρ(PO4)-3= 25 mg/l;
ρ(SO4)-2 = 100 mg/l.

Pipette 200 ml of element stock solution (Ca), 300 ml of anion stock

solution (Cl-), 25 ml of anion stock solution (PO4)-3 and 100 ml of
anion stock solution (SO4) -2 to a 1 000 ml volumetric flask.
Add 10 ml of nitric acid.
Bring to volume with water and transfer to a suitable storage bottle.
Sample Preparation Sampling
Due to the extremely high requirements concerning purity in trace and
ultra trace analysis any impurity shall be avoided.
The mass concentrations of the elements may change rather rapidly after
sampling due to adsorption or desorption effects. This is of special
importance, for example in the case of Ag, As, B, Se and Sn. The choice
of the container material depends on the mass concentration of the
elements to be determined.

For the determination of the dissolved fraction of the elements, filter the
sample through a membrane filter, nominal pore size 0.45 μm. Every
batch of membrane filters shall be tested for impurities. Use several
portions of the sample to rinse the filter assembly, discard and then collect
the required volume of filtrate.

25
 Add 0.5 ml of nitric acid per 100 ml of sample. Ensure that the pH is less
than 2; otherwise, add nitric acid as required.

In the case of determination of elements forming compounds that tend to

be hydrolysed, for example Sb, Sn,W or Zr, add to an additional sample
1.0 ml of hydrochloric acid per 100 ml of water. Ensure that the pHis less
than 1; otherwise, add more hydrochloric acid as required.

Sample pre-treatment

1) Determination of the mass concentration of dissolved elements

without digestion
Continue according to sampling procedure,using the acidified filtrate
specified in sampling. If experience has shown that no significant
amounts of particles occur, the filtration may be omitted. Those samples
shall be colourless and shall have a turbidity <1.5 FNU
(formazinenephelometric unit).

2) Determination of the total mass concentration after digestion

The mass concentration determined according to this clause does not in all
cases represent the total mass concentration. Instead, only the portion that
is determinable according to the distinct digestion for a given element
composition will be analyzed.
Some elements and their respective compounds (for example, silicates
and aluminum oxide) will be dissolved incompletely using this procedure.

For the determination of tin, the following digestion may be used:

a) Add 0.5 ml of sulfuric acid and 0.5 ml of hydrogen peroxide to 50

ml of the homogenized water sample.
b) Evaporate the mixture until SO3vapors is formed.
c) In case of incomplete digestion, add a small portion of water after
cooling, add hydrogen peroxide once more and repeat the treatment.
d) Dissolve the residue in diluted hydrochloric acid and adjust the
volume to 50 ml with water.
e) Treat a blank in the same way.

Special digestion methods may be necessary if Sb, W or Zr is to be

determined.
If experience has shown that the elements will be recovered quantitatively
without decomposition, the digestion may be omitted.
Method of analysis 1) General-
In ICP-MS methods, the relationship between measured count rates
and mass concentrations of an element is known to be linear over
several orders of magnitude. Therefore, linear calibration curves may

26
 be used for quantification.

About 30 min prior to measurement, adjust the instrument to working

condition.
Before each series of measurement the sensitivity and the stability of
the system should be checked using the optimization solution.

Adjust the instrument with the aid of the optimization solution to

minimize interfering effects (for example oxide formation, formation
of doubly charged ions) allowing sufficient sensitivity.

Define the rinsing times depending on the length of the flow; in the
case of large variations in mass concentrations in the measuring
solutions, allow for longer rinsing periods.

The use of a reference-element solution is recommended. Add the

reference-element solution to the matrix solution, to all multi-element
calibration solutions, to the blank calibration solutions, and to all
measuring solutions. The mass concentration of the reference-elements

50 μg/l is often suitable.

ICP-MS has excellent multi-element capability. The sensitivity of

determination depends on a number of parameters (nebulizer flow,
radiofrequency power, lens voltage, lens voltage mode, etc.). The
optimal instrument settings cannot be achieved for all elements
simultaneously.

2)Calibration of the ICP-MS system

When the analytical system is first evaluated, and at intervals
afterwards, establish a calibration curve for each element to be
determined using at least five measuring points (for example, the blank
calibration solution and four calibration solutions).
For work on a daily basis, one blank solution and one to two
calibration solutions are enough but check the validity of the
calibration curve with a certified reference sample, a standard sample,
or a suitable internal control sample.
Typically proceed as follows:
Prepare and measure the blank calibration solutions and the multi-
element calibration solutions.
According to the manufacturer's instruction, set up a calibration graph.
Each reference point should be themean of at least two replicates.
Take into account possible discrepancies in the isotope composition
between the calibration solutions and themeasuring solutions (for
example relevant for Li, Pb, U).

27
 3) Measurement of the matrix solution for evaluation of the
correction factors
In order to evaluate and to update the correction factors, measure the
matrix solutions at regular intervals within a measuring cycle.

4) Measurement of the samples

After establishing the calibration curves, measure the blanks and the
samples.
Within sufficient small intervals (for example, every ten samples)
check the accuracy of at least one certified reference sample or one
standard sample or one internal control sample. If necessary, re-
calibrate.
Some elements (for example Ag, B, Be, Li, Th) are rinsed very slowly
from the sample inlet system. After high count rates, these memory
effects shall be checked by measuring a blank calibration solution.
Calculation with units of Calculation
expression The mass concentrations for each element are determined with the aid of
the instrument software. Carry out the following single steps for each
element.

a) Correct the count rates according to the respective equations as

below:Examples for suitable isotopes with their relative
atomicmasses and equations for correction

Element Recommended isotope and inter-element

correction
As 75As -3.127 (77Se- 0.815 82Se) or
75As -3.127 (77Se + 0.322 0 78Se)

Ba 138Ba -0.0009008 139La - 0.002 825 140Ce

Cd 114Cd-0.02684 118Sn
Ge 74Ge -0.1385 82Se
In 115In -0.01486 118Sn
Mo 98Mo -0.1106 101Ru
Ni 58Ni -0.04825 54Fe
Pb 208Pb + 207Pb + 206Pb
Se 82Se- 1.009 83Kr
Sn 120Sn−- 0.013 44 125Te
V 51V - 3.127 (53Cr -0.113 4 52Cr)
W 184W -0,001 242 189Os

b) Make allowance for the count rates from the blank calibration,
calibration and measuring solutions, and relate to the count rates of the
reference-elements. Determine the slope and the intercept on the ordinate.

28
 c) Determine the mass concentrations of samples with the aid of the count
rates and the calibration graphs.
d) Correct the results taking into account the mass concentrations from the
blank calibration solutions and incorporate all dilution steps in the
calculation. If the sample is digested a correction for the procedure blank
shall be used if appropriate (digestion blank solution).

Report the results to as many significant figures as are acceptable

according to the precision of the measuring values.
Examples- Copper (Cu) 0.142 mg/l, Cadmium (Cd) 0.50 μg/l
Inference
(Qualitative Analysis)
Reference ISO 17294 Water quality — Application of inductively coupled plasma
mass spectrometry (ICP-MS)
Approved by Scientific Panel on Methods of Sampling and Analysis

29
 Method for the analysis of Arsenic, Cadmium, Mercury, and Lead in
Foods by Pressure Digestion and Inductively Coupled Plasma-Mass
Spectrometry

Method No. FSSAI 09.004:2024 Revision No. & Date 0.0

Scope Applicable to the determination of As, Cd, Hg, and Pb in a variety of
foods by pressure digestion and inductively coupled plasma-mass
spectrometry (ICP-MS). Method is capable of determining As, Cd, Pb,
and Hg at or above 0.06, 0.03, 0.04, and 0.09 mg/kg dry matter,
respectively.
Caution
1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
3) Reagents should be regarded as potential health hazards and exposure
to these materials should be minimized. Follow universal precautions.
Wear gloves, a lab coat, and safety glasses while handling reagent.
4) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
Principle Foodstuffs are mineralized (digested) in closed vessels by nitric acid at
elevated temperature and pressure by conventional or microwave-assisted
heating. The mineralized sample is diluted with water to a defined volume
to produce the test solution. Test samples may be either dry or wet. Test
samples may be dried and results corrected for moisture.

The test solution, obtained by pressure digestion, is transferred to the

sample introduction system of the ICP-MS instrument and nebulized, and
the aerosol is transferred to high frequency inductively coupled argon
plasma. The high temperature of the plasma is used to dry the aerosol and
to atomize and ionize the elements. The ions are extracted from the
plasma by a set of sampler and skimmer cones and transferred to a mass
spectrometer by vacuum, where the ions are separated by their
mass/charge ratio (m/z) and determined by a pulse-count and/or analog
detector.
Apparatus/Instruments 1) Pressure digestion—Commercially available microwave digestion
system or high-pressure asher for acid digestion in an acid-resistant
sealed vessel with a low level of contamination. Capable of digestions
by conventional or microwave-assisted heating in a sealed vessel in a

30
 pressure container.
2) Inductively coupled plasma-Mass spectrometer (ICP-MS)—Mass
spectrometer with inductively coupled argon plasma operating in a
mass range from 5–240 amu. Mass spectrometers with additional
reaction or collision cells may be used to reduce the influence of
polyatomic ions. An ICP-MS instrument having a nebulizing system
with a low pulsation peristaltic pump should be equipped with a mass
flow controller for the nebulizer gas.
Materials and Reagents 1) Nitric acid.—Not less than 65%, with a density of ca 1.4 g/mL.
2) Hydrogen peroxide (H2O2).—30%.
3) Water.—Specific resistance >18 megohm-cm.
4) Element stock solutions- Commercially available single element or
multi-element standards with a concentration of 1000 mg/L are
recommended.
Preparation of Reagents 1) Diluted Hg stock solution—Hg = 10 mg/L, prepared by dilution of 1
mL Hg and 1 mL nitric acid with water to the mark of a 100 mL
volumetric flask.

2) Diluted multi-element stock solution—The concentration levels of

the elements in the diluted multi-element stock solution may be
chosen with reference to the type of samples to be analyzed. The
following descriptions are given as an example: As = 20 mg/L, and
Cd and Pb = 10 mg/L. Pipet 2 mL As stock solution and 1 mL of the
single element standards Cd and Pb each in a 100 mL volumetric
flask, add 1 mL nitric acid, dilute with water to the mark, and transfer
the solution into a suitable vessel.

3) Multi-element calibration stock solution—According to the

example given, the multi-element calibration stock solution contains
100 μg As/L and 50 μg/L for Cd, Hg, and Pb. Pipet 0.5 mL of diluted
Hg stock solution and 0.5 mL of the diluted multi-element stock
solution into a 100 mL volumetric flask, add 1 mL nitric acid, dilute
with water to the mark, and transfer the solution into a suitable vessel
(PFA or quartz is recommended).

4) Calibration solutions—For calibration of the instrument a set of at

least three different concentrations are used (in addition to the
standard reagent blank). The concentration range should be chosen
with respect to the concentrations expected and with respect to the
linear dynamic range. It is important that the concentration of nitric

31
 acid in the sample solutions and the calibration solutions are
approximately the same. The calibration solutions should be prepared
freshly before use.

5) Standard reagent blank—Standard reagent blank contains water

and the same amount of acid used in the calibration stock solution.

6) Internal standard stock solution—Rh and Lu with a concentration

of 1000 mg/L is recommended. Alternatively, other internal standards
may also be used. Au is used to stabilize Hg in the solution and to
reduce memory effects. The internal standards should cover the mass
range used for determination of the elements. Their concentrations in
the test solutions should be negligible.

7) Diluted internal standard stock solution—The concentration of the

diluted internal standard solution should be high enough to give a
sufficient signal intensity. For example, Au, Rh, and Lu at 5 mg/L
can be used. Pipet 0.5 mL of Au, Rh, and Lu internal standard stock
solution each into a 100 mL flask, add 1 mL nitric acid, dilute to
volume with water, and transfer the solution into a suitable vessel.

8) Optimization solution—The optimization solution is used for check

and optimizing procedures during set up of the ICP-MS instrument. It
is used for mass calibration purposes and for adjustment of maximum
sensitivity at low rates of oxides and doubly charged ions. The
optimizing solution should contain elements that cover the whole
mass range giving a high rate of oxides and double charged ions. The
solutions recommended by the manufacturer of the ICP-MS
instrument may be used. A solution containing, e.g., Y, Rh, Ce, and
Pb is suitable for those purposes. The concentration of these elements
should be chosen in order to achieve a count rate of 10 000–100 000
cps.
Sample Preparation 1) Equipment which does not impart any or least possible
contamination particularly with respect to the analytes of the interest
is used to homogenize the sample.
Caution: Digestion of carbon-rich materials (e.g., carbohydrates,
fats, oils) can result in explosions.
2) Moisture content (optional)—To avoid possible losses of volatile
elements such as As and Hg, the determination of moisture content

32
 should be done on separate homogenized test portions rather than on
test portions used for analysis. Determination of optimal drying
temperatures and times are needed to avoid mass loss due to loss of
volatile oils. Where previous drying studies have been conducted,
recommended temperatures and drying times can be used. For oven
drying, temperatures may range from 80°C to 110°C until constant
mass is reached. Alternatively samples can be dried over
Mg(ClO4)2 in a sealed desiccators until constant mass is reached.
The drying factor necessary to convert the mass of the stored
material to a dry-mass basis should be determined at each use to
account for changes in mass due to the exposure of the material to
the laboratory atmosphere.
3) Food samples are digested in sealed pressure digestion vessels. The
sample mass is chosen to match the capacity of the digestion vessel
following the manufacturer’s instructions which may also limit the
carbon content to be digested. The test sample portion and the
appropriate amounts of nitric acid and hydrogen peroxide are placed
in the digestion vessel. The vessel is secured in the pressure digester
and the temperature/pressure program implemented.

Microwave-assisted wet digestion:

(a) Weigh approximately 0.20 g dry weight sample material into the
digestion container.
(b) Each digestion series must contain two reagent blanks (nitric acid
and hydrogen peroxide without sample materials). Include at least
one certified reference material corresponding to the amount
expected in the samples, in order to reveal systematic errors.
(c) Digestion programs and amounts of acids will vary with different
digestion systems. Add 2 mL concentrated nitric acid and 0.5 mL
hydrogen peroxide to each container.
(d) Seal the containers in the capping station. Place carousel with the
digestion containers in the microwave oven and start the digestion
program.
(e) The digested solution is diluted by water to a known volume (test
solution).
Method of analysis 1) Preparation of calibration solutions and test solutions for ICP-
MS measurement—All solutions to be measured by ICP-MS
during routine runs should contain one or a set of internal standards.
The concentration of the internal standard(s) must be equal in all of

33
 the solutions. For the determination of Hg, Au must be added in
order to stabilize the Hg and minimize memory effects in the tubing
and during nebulization. The test solution obtained by pressure
digestion should be analyzed after dilution to a known volume.
Example: Pipet exactly 10 mL of standard reagent blank or
calibration solution to a vessel; add 0.1 mL diluted internal standard
stock solution and mix. Pipet exactly 2 mL of test solution to a
vessel; add exactly 8 mL water and 0.1 mL diluted internal standard
stock solution and mix. Every solution contains ca 10 μg/L of the
internal standard Rh. The internal standard solution may also be
added online by a different channel on the peristaltic pump used for
the analytes. Adjust the concentration of the internal standard
solution and the pump flow rate in order to achieve a concentration
of the internal standard of ca 50 μg/L.
2) Calibration of the ICP-MS instrument—For calibration purposes,
a minimum of three different concentrations must be used. Measure
the standard reagent blank and then the calibration solutions.
According to the instrument manual, calculate the calibration
function. Different isotope ratios between calibration solutions and
test solutions should be taken into account, if necessary.
3) Analyses of test solutions—After calibration of the instrument, the
test solutions can be analyzed. The samples obtained by pressure
digestion should be diluted before measurement in order to avoid
interference by high concentrations of matrix elements. If the final
volume of the digested solution is 20–30 mL, a dilution by a factor
of 5–10 is recommended for the ICP-MS measurement. Within
suitable short intervals (e.g., after 5 or 10 samples), the blank
solution and one calibration solution should be checked regularly.
The recovery of the calibration solution should range within 10%.
For high concentrations of Hg, prolonged washout times have to be
applied. The blank level for Hg should be checked regularly in order
to detect any memory or washout effects. The system should be
tested for washout times using the highest calibration standard.
4) Control for matrix effects—The amount of matrix present in the
test solution to be analyzed may create more or less significant
matrix effects compared to pure multi-element standards. To check
for matrix effects, a known amount of the multi-element standard is
added to the test solution.
Example: Pipet exactly 2 mL test sample into a sample vessel, and

34
 add exactly 7 mL water and 1 mL Calibration Solution 3. Then add
0.1 mL internal standard stock solution and mix. The non-added
sample is prepared in the same way by using 1 mL water instead of
the calibration solution. The concentrations found by addition of the
standard should not exceed 10% of the added concentration. In case
of greater differences, the matrix effects must be compensated by a
standard addition calibration.
5) Standard addition calibration—A standard addition calibration
should consist of at least three points, of which two are standard
additions. The concentration of the highest standard should be three
to five times the concentration in the sample solution. The
concentration of the lower standard should be half of the highest
standard, i.e., 100, 200, and 400% of the initial concentration in the
test sample. The non-spiked test solution is used as the lowest level
in the calibration curve. The linear regression through these points
crosses the negative concentration axis. The absolute value of this
point is the concentration of the element in the sample solution.
Example: For a test solution containing ca 0.5 μg Cd/L, pipet into 4
different sample vessels exactly 2 mL of each test sample. To the
first sample vessel, add exactly 8 mL water (= non-spiked test
solution). To the second sample vessel, add exactly 7.5 mL water
and 0.5 mL Calibration Solution 3 (= Sample Spike 1, with an added
concentration of 0.5 μg Cd/L). To the third sample vessel, add
exactly 7 mL water and 1 mL Calibration Solution 3 (= Sample
Spike 2, with an added concentration of 1 μg Cd/L). To the fourth
sample vessel, add exactly 6 mL water and 2 mL Calibration
Solution 3 (= Sample Spike 3, with an added concentration of 2 μg
Cd/L).
Calculation with units of Calculation of the concentration is generally done automatically by the
expression software of the ICP-MS instrument. The following steps are performed
for each element: The count rates are corrected according to the
correction functions chosen, the count rates are measured in the
standard reagent blank, and calibration and test solutions are normalized
on the count rates of the internal standard. The calibration function is
then calculated. By the use of the count rates, the calibration function
and the dilution factor of the concentrations of the elements are
calculated. The content, W, as mass fraction, of the element to be
determined in mg/kg of sample is calculated using the following
equation:

35
 axVxF
W=
m x 1000
where a is the content (μg/L) of the element in the test solution, V is the
volume (mL) of the digestion solution after being made up to volume, F
is the dilution factor of the test solution, and m is the mass of the test
portion (g). Report moisture content if test samples were dried and
indicate mass fraction (W) as dry matter. Alternatively, correct dry
matter result for moisture content.
Inference
(Qualitative Analysis)
Reference AOAC Official Method 2013.06
Approved by Scientific Panel on Methods of Sampling and Analysis

36
 Method for the analysis of Chromium, Selenium, and Molybdenum
in Infant Foods and Adult Nutritional Food Products by
Inductively Coupled Plasma-Mass Spectrometry

Method No. FSSAI 09.005:2024 Revision No. & Date 0.0

Scope Applicable to the determination of Cr, Se, and Mo in Infant Formula and
Adult Nutritional Products by pressure digestion and inductively coupled
plasma-mass spectrometry (ICP-MS). Method is capable of determining
Cr, Se, and Mo at or above 0.06, 0.03, and 0.09 mg/kg dry matter,
respectively.
Caution
1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Microwave operation involves hot pressurized acid solution. Use
appropriate face protection and laboratory clothing.
3) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
4) Reagents should be regarded as potential health hazards and exposure
to these materials should be minimized. Use normal laboratory safety
precautions (laboratory coats and safety glasses with side shields)
when handling concentrated acids, bases, and organic solvents.
Additional protections such as face shields, neoprene gloves, and
aprons should be used where splashing may occur.
5) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
Principle Test portion is heated with nitric acid in a closed vessel microwave
digestion system at 200°C. Digested test solution, or an appropriate
dilution, is presented to the inductively coupled plasma-mass
spectrometer (ICP-MS) instrument standardized with acid matched
standard calibrant solutions. An ionization buffer (potassium) is used to
minimize easily ionizable element (EIE) effects, methanol is added to
normalize the carbon content, and nickel and tellurium are used as
internal standards.
Apparatus/Instruments 1) Microwave—Commercial microwave designed for laboratory use
at 0–300°C, with closed vessel system and controlled temperature
ramping capability. It is recommended that the vessel design be
selected that will withstand the maximum possible pressure, since
organic material, and also carbonates if not given sufficient time to
predigest, will generate significant pressure during digestion.

37
 (Vessels can reach 700 psi or more on occasion.) Vessels must be
designed to operate with only 6 mL solution volume, or the volume
must be adjusted accordingly.
2) ICP-Mass Spectrometer—With collision reaction cells (CRCs).
3) Various plastic ware and pipets.
Materials and Reagents 1) Laboratory water—Use 18 MΩ water throughout for dilution.
2) Concentrated nitric acid (HNO3)—65–70% trace metal-grade
HNO3 throughout.
3) Hydrogen peroxide—30% ACS reagent grade.
4) Methanol—99.99% analytical reagent grade for matrix matching.
5) Potassium—10 000 mg/L in nitric acid for matrix matching.
Preparation of Reagents 1) 2 mg/L Cr and Mo and 1 mg/L Se multi-element stock standard
solution in nitric acid—High-Purity Standards, or equivalent.

2) 5 mg/L Ni and Te multi-element stock standard solution in nitric

acid.—High-Purity Standards, or equivalent.

3) Standard preparation—

(a) Prepare intermediate standards from commercial stock

standards at 40 ng/mL Cr and Mo, and 20 ng/mL Se. Custom-
blended multi-element stock standard in HNO3 is acceptable.
(b) Prepare a minimum of three multi-element working standards
containing 0.8, 4.0, and 20 ng/mL Cr and Mo and 0.4, 2.0, and
10 ng/mL Se, plus blank, with both Ni and Te internal
standard, in HNO3. Ni is used as the internal standard for both
Cr and Mo, and Te must be used for Se.
Sample Preparation 1) Prepare powder samples by reconstituting approximately 25 g
sample in 200 ml warm laboratory water (60°C).
2) Accurately weigh approximately 1.8 g reconstituted test portion into
the digestion vessel. This represents approximately 0.2 g original
powder sample.
3) Fluid samples may be prepared by accurately weighing
approximately 1 g test portion weighed directly into the digestion
vessel after mixing.
4) Microwave digestion-
(a) Add 0.5 mL 5000 ng/mL Ni and Te internal standard solution
and 5 mL trace metal-grade HNO3 followed by 2 mL H2O2 to
the microwave digestion vessels.
(b) Seal vessels according to manufacturer’s directions and place
in microwave.
(c) Ramp temperature from ambient to 180°C in 20 min, and hold
for 20 min in stage 1. In stage 2, the microwave will

38
 automatically ramp to 200°C in 20 min, and hold for 20 min.
(d) Cool vessels according to manufacturer’s directions,
approximately 20 min.
(e) Slowly open the microwave vessels, venting the brownish
nitrogen dioxide gases.
(f) Add 1 mL H2O2 and redigest samples by ramping the
temperature from ambient to 180°C in 15 min. Hold at 180°C
for 15 min and cool for 20 min.

5) Preparation of test solution—Add approximately 20 mL

laboratory water to the contents of the vessel with the digested
samples and transfer to a 50 mL sample vial. Rinse the vessel and
transfer the rinsate into the sample vial. Add 0.5 mL methanol to the
sample vial and dilute to 50 mL with laboratory water.
Method of analysis 1) Analyze test solutions using an ICP-MS instrument standardized
with standard solutions. Ni is used as the internal standard for both
Cr and Mo (helium mode), and Te must be used for Se (hydrogen
mode).
2) Analyze a 4 ng/mL Cr and Mo, and 2 ng/mL Se working standard or
other suitable quality control solution every 10 test portions to
monitor for instrument drift and linearity (result 100 ± within 5% of
nominal).
3) The inclusion of a method blank (run as a sample), a duplicate
sample [relative percent difference (RPD) ≤ within 10%], and
known reference materials serving as control samples (recovery
check within control limits) are considered mandatory for good
method performance. If any of these QC checks fails, results should
be considered invalid.
Calculation with units of Sample concentrations were automatically calculated by the ChemStation
expression software using a nonweighted least-squares linear regression calibration
analysis to produce a best-fit line:
Y = ax + blank
The analyte concentration in the sample was then calculated:
y- blank
x= x DF
a
where
x = analyte concentration (ng/g);
y = sample response ratio (ng/mL), which is the measured count of
each analyte’s standard solution data point in the calibration curve

39
 divided by the ratio of the counts/concentration of the internal
standard at the same level;
blank = blank standard solution (ng/mL), which is the measured
count of the blank standard solution data point in the calibration
curve divided by the ratio of the counts/concentration of the
internal standard at the same level as the blank standard solution;
a = slope of the calibration curve;
DF = dilution factor of the sample solution divided by sample weight
(mL/g).
Inference
(Qualitative Analysis)
Reference AOAC Official Method 2011.19

Approved by Scientific Panel on Methods of Sampling and Analysis

40
 Method for the analysis of Total Iodine in Infant Foods and
Adult/Pediatric Nutritional foods by Inductively Coupled Plasma–
Mass Spectrometry

Method No. FSSAI 09.006:2024 Revision No. & Date 0.0

Scope Applicable for the determination of total iodine in infant formula and
adult nutritional products by ICP/MS using microwave oven closed-vessel
acid digestion.
Caution
1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Oven and microwave digestion procedures involve moderately
elevated temperatures. Carefully remove samples and allow cooling
before removing the lids from the digestion vessels.
3) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
4) The method involves the use of strong bases and concentrated acids.
Avoid spills, inhalation, and exposure to human tissues. Use normal
laboratory safety precautions (laboratory coats and safety glasses with
side shields) when handling concentrated acids, bases, and organic
solvents. Additional protections such as face shields, neoprene gloves,
and aprons should be used where splashing may occur.
5) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
Principle Test portion is digested with KOH solution in an oven at 105 ± 5°C in
a closed vessel until the dissolution of iodine is complete. Mix the
digested test solution with stabilizer concentrate. Filter the sample
through a 1 µm membrane filter. This sample solution or an
appropriate dilution, is presented to the inductively to inductively
coupled plasma-mass spectrometer (ICP-MS) instrument standardized
with matched standard calibrate solutions.
Apparatus/Instruments 1) Polypropylene (PP) tubes.—Assorted sizes, use as received.
2) Oven (i.e., warming/drying oven).
3) Open-vessel microwave digestion unit (optional).
4) Analytical and top-loader balances.—Sensitive to 0.0001 and 0.01 g,
respectively.
5) ICP-MS system.
6) Auto sampler for ICP-MS.
7) Adjustable (electronic or manual) volumetric pipets and pipet tips.
8) Re-pipet volumetric dispenser—Adjustable volume.

41
 9) Polypropylene or Teflon bottles for storage of reagents.
10) Disposable plastic syringes.
11) Syringe filters with 1 μm membrane.

Note: All laboratory plasticware should be single-use wheneverpossible.

If reuse is necessary, wash using 10% nitric acid, thenrinse
thoroughly with purified water prior to use.
Materials and Reagents 1) KOH (KOH) pellets, certified ACS—KOH may contribute
background levels of iodine.
2) KOH solution—50% (w/v).
3) Ammonium hydroxide (NH4OH)—Certified ACS.
4) Sodium thiosulfate (Na2S2O3)—99.99+% metal basis.
5) Surfactant (i.e., Triton® X-100).
6) Nitric acid (HNO3)—High purity.
7) Perchloric acid (HClO4)—High purity.
8) Purified water—18 MΩ/cm.
Preparation of Reagents Iodine stock standard solutions—Certified ICP-MS or ICP grade
single- or multi-element standard solutions (or other certified reference
materials; CRM) are used to prepare calibration, calibration verification
standards, internal standards, and spiking solutions.

Likely choices for use as internal standards for iodine analysis are
praseodymium (Pr), samarium (Sm), tellurium (Te), and rhodium (Rh).
Concentrations used for analysis are 30.0 ppb Pr, Sm, Rh, and 500 ppb
Te. The internal standard solution reagent’s concentration is 2% HNO3,
0.1% HClO4, 0.01% Triton X-100, 0.25% KOH, 0.1% NH4OH, and
0.01% Na2S2O3 in purified water.
1) 5% KOH solution.—Dissolve 25 g KOH pellets in an appropriate
amount of purified water, then dilute to 500 mL with purified water.
Store at room temperature. Reagent expires 6 months after
preparation date. Alternatively, dilute 50 mL 50% (w/v) KOH
solution to a final volume of 500 mL with purified water. Store at
room temperature. Reagent expires 6 months after preparation date.
2) 50% KOH solution.—Dissolve 250 g KOH pellets in an appropriate
amount of purified water, then dilute to 500 mL with purified water.
Store at room temperature. Reagent expires 6 months after
preparation date.
Note: Use caution when preparing this solution as a significant
amount of heat is generated.
3) Stabilizer concentrates—Dissolve 5 g Na2S2O3 in an appropriate
amount of purified water, add 50 mL NH4OH, then dilute to 500 mL
with purified water. The resulting concentration
is 10% NH4OH and 1% Na2S2O3 in purified water. Store at room
temperature. Reagent expires 6 months after preparation date.
4) Wash solution (rinse)—Dissolve 2 g Triton X-100 in an

42
 appropriate amount of purified water, add 20 mL NH4OH, then
dilute to 2 L with purified water. The resulting concentration is 1%
NH4OH and 0.1% Triton X-100 in purified water. Store at room
temperature.
5) Diluent—Dissolve 10 g KOH pellets and 0.4 g of Na2S2O3 in an
appropriate amount of purified water, add 4 mL NH4OH, then dilute
to 2000 mL with purified water. Store at room temperature.
Alternatively for a smaller volume, dilute 50 mL 5% KOH and 10
mL stabilizer concentrate to 500 mL with purified water. Store at
room temperature.
Note: The resulting concentration for both preparations is
0.5%KOH, 0.2% NH4OH, and 0.02% Na2S2O3 in purified water.
6) Conditioning solution—Prepare by aliquoting 25 mL 5% KOH
(2.5 mL 50% KOH) solution, then diluting to 250 mL with purified
water. This solution is used to prepare the instrument for analysis.
The resulting concentration is 0.5% KOH. Store at room
temperature.
7) Carrier solution—Equivalent to the wash solution. The carrier
solution is used to deliver the sample solution to the nebulizer
through the ICP-MS auto sampler introduction system. The carrier
solution is introduced via a peristaltic pump using black/black two
stop
polyvinyl chloride pump tubing (0.76 mm id). Store at room
temperature.
Note: All above reagent expires in 6 months after preparation date.
Sample Preparation 1) Oven digestion (preferred)—Note: The following oven digestion
procedure is for a final volume of 100 mL. Samples expected to
contain levels of iodine below 10 000 μg/kg may be digested using the
5% KOH solution. However, if samples are expected to contain >10
000 μg/kg iodine and are anticipated to be detectable after an
appropriate dilution, the 50% KOH solution may be used.
Vitamin/mineral dietary supplements or premixes or other certain
matrixes should be digested using only the 50% KOH solution.
For the testing of vitamin/mineral tablets or premixes, it is
recommended (due to potential homogeneity issues) that a
reconstitution be performed. Unless a specific reconstitution
procedure is required, use the following reconstitution procedure as a
guide.
Suggested reconstitution procedure—
(a) Accurately weigh approximately 5.00 g of sample into an
appropriate vessel (150 mL or 250 mL beaker) and record the
sample weight. Without zeroing the balance, add water to make
approximately 100 g. Record the sample + water weight. Place a
stir bar in the mixture and stir on a stir plate to form a
homogenous slurry/suspension.

43
 While stirring, weigh 5–10 g of the slurry/suspension into an
appropriate digestion vessel, add approximately 10 mL water, then
proceed with the addition of the KOH as stated below.
(b) Accurately weigh or aliquot an appropriate amount (0.2500 to 2.50
g or 0.50 to 10 mL) of sample into a labeled 100 mL digestion
vessel. Add 20 mL purified water to the vessel.
Accurately weigh an appropriate amount (0.2500 to 1.00 g) of an
appropriate CRM, i.e., National Institute of Standards and
Technology Standard Reference Material (NIST SRM) 1549 or
3280, if applicable, in the same manner as the samples. SRM 1549
may be digested using either 5 or 50% KOH solution. SRM 3280
should be digested using only the 50% KOH solution.
(c) Designate at least one digestion vessel as the digest blank. The
digestion blank(s) should be treated in the same manner as the
samples. If both the 5 and 50% KOH solutions will be used,
prepare at least one blank with each concentration. Place an
aliquot of spiking solution (if applicable) into an appropriately
labeled digestion vessel.
(d) Add either 10 mL 5% KOH solution or 10 mL 50% KOH solution
to each digestion vessel.
Note: If values well below 10 000 μg/kg are anticipated, add 5 mL
5% KOH solution.
(e) Dilute to 50 mL. Seal the vessels and swirl or use a vortex
apparatus to mix. Avoid inverting as this may allow sample to
adhere to the inner walls of the vessel above the level of the
digestion solution. Digest samples in an oven set to maintain 105
± 5°C until the dissolution of iodine is complete, approximately 1
h.
(f) After removal from the oven, allow samples to cool first, then
add2 mL stabilizer concentrate and bring to volume with purified
water.
Note: If the final volume will be 50 mL, add 1 mL stabilizer
concentrate.
(g) Cap the vessels, then invert to mix thoroughly. Filter the sample
solution by filling a disposable syringe with the digested sample
solution, attach a 1 μm membrane filter, then filter an adequate
amount (i.e., several milliliters) into appropriate vessel (i.e.,15 mL
PP centrifuge tube) to be used for analysis. Store samples at
ambient temperature.

2) Open vessel microwave digestion (optional)-

(a) Accurately weigh approximately 5.00 g of sample into an
appropriate vessel (150 mL or 250 mL beaker) and record the
sample weight. Without zeroing the balance, add water to make
approximately 100 g. Record the sample + water weight. Place a

44
 stir bar in the mixture and stir on a stir plate to form a
homogenous slurry/suspension.
While stirring, weigh 5–10 g of the slurry/suspension into an
appropriate digestion vessel, weigh 5 g of the slurry/suspension,
and do not add additional water. Proceed with the addition of
KOH as described below.
(b) Accurately weigh or aliquot an appropriate amount (0.2500 to 1.00
g or 0.50 to 2 mL) of sample into a labeled microwave digestion
vessel already contains 5 mL purified water.
(c) Designate at least one digestion vessel as the digest blank. The
digestion blank(s) should be treated in the same manner as the
samples. If both the 5 and 50% KOH solutions will be used,
prepare at least one blank with each concentration. Place an
aliquot of spiking solution (if applicable) into an appropriately
labeled microwave digestion vessel.
(d) Add either 5 mL 5% KOH solution or 5 mL 50% KOH solution to
each digestion vessel.

Note: If values well below 500 μg/kg are anticipated, add 5 mL of 5%

KOH solution.
(e) Seal the vessels and swirl or use a vortex apparatus to mix. Avoid
inverting as this may allow sample to adhere to the inner walls of
the vessel above the level of the digestion solution. Place the
digestion vessels into the carousel of the open-vessel microwave
digestion unit. If less than the maximum capacity is to be digested,
distribute the vessels evenly throughout the carousel. Digest the
samples in the microwave until the dissolution of iodine is
complete. The vessel caps should be loosened slightly (from fully
tightened) during the digestion procedure.
(f) After removal from the oven, allow sample to cool first, then add
1 mL stabilizer concentrate and bring to volume with purified
water. Cap the vessels, and then invert to mix thoroughly.
(g) Filter the sample solution by filling a disposable syringe with the
digested sample solution, attach a 1 μm membrane filter, then
filter an adequate amount (i.e., several milliliters) into appropriate
vessel (i.e., 15 mL PP centrifuge tube) to be used for analysis.
Store samples at ambient temperature.
Method of analysis The digested samples are analyzed directly or diluted so that the iodine
concentration will fall within the calibration range.
1) Samples digested with 50% KOH solution must be diluted 1 to 10
mL to achieve the desired final concentration of 0.5% KOH.
2) Aliquot 1 mL of the filtrate into an appropriate vessel (i.e., 15 mL
PP centrifuge tube), add 0.18 mL stabilizer concentrate, then
dilute to 10 mL with purified water.
3) If samples digested with 50% KOH solution need more than a 1 to

45
 10 mL dilution to obtain a reading on the calibration curve, an
additional dilution must be prepared from the original 1 to 10 mL
dilution.
4) Aliquot the desired amount into an appropriate vessel (i.e., 15 or
50 mL PP centrifuge tube), then dilute to volume with diluent.
5) Analyze conditioning solution while concomitantly introducing
internal standard solution on-line through a mixing block until
conditioned (approximately 1 h). The internal standard solution is
introduced via a peristaltic pump using orange/green two-stop
PVC pump tubing (0.38 mm id). After conditioning, begin to
aspirate carrier solution while continuing to add internal standard.
Analyze samples using ICP-MS.

Isotope selection and interferences- The internal standards listed below

can use for method development:
Analyte Mass, amu
Iodine 126.900
Praseodymium 140.907
Samarium 146.915
Rhodium 102.906
Tellurium 146.915

Calculation with units of Calculation of the concentration is done automatically by the software of
expression the ICP-MS instrument.
Inference
(Qualitative Analysis)
Reference AOAC Official Method 2012.15
Approved by Scientific Panel on Methods of Sampling and Analysis

46
 Method for the Determination of Methyl Mercury and Total Mercury
in Seafood by High Performance Liquid Chromatographic-
Inductively Coupled Plasma–Mass Spectrometry

Method No. FSSAI 09.007:2024 Revision No. & Date 0.0

Scope This method describes procedures for using high performance liquid
chromatography (HPLC) and inductively couple plasma-mass
spectrometry (ICP-MS) to determine methyl mercury and total mercury in
seafood. Total mercury in this method is calculated as the sum of
inorganic and methyl mercury determined in analytical solution.
Analytical parameters LOD (μg/kg) LOQ (μg/kg)
Methyl mercury 3.8 28
Total Mercury 6.5 47
Caution
1) Use fume hood and wear full personal laboratory protective clothing,
gloves, and appropriate eye protection (safety glasses) when using
glassware and preparing standards or test portions with acid solutions.
2) Inductively coupled plasmas should only be viewed with proper eye
protection from ultraviolet emissions.
3) Reagents should be regarded as potential health hazards and exposure
to these materials should be minimized as much as possible. Use
normal laboratory safety precautions (laboratory coats and safety
glasses with side shields) when handling organic solvents. Additional
protections such as face shields, neoprene gloves, and aprons should
be used where splashing may occur.
4) Exercise caution when handling and dispensing concentrated acids.
Always add acid to water. Acids are caustic chemicals that are capable
of causing severe eye and skin damage. If acids or bases come in
contact with any part of the body, quickly wash the affected area with
copious quantities of water for at least 15 minutes.
Principle Hg species are isolated from 0.5 g non-dried, finely comminuted
seafood or 0.2 g dried reference material by extracting with 50 mL
aqueous solution of 1% (w/v) L-cysteine.HCl.H2O for 120 minutes at
60 °C. The seafood-cysteine mixture is cooled to room temperature
and filtered to remove particles > 0.45 μm diameter. Fifty μL portions
of filtered extract are injected onto a C-18 column where Hg species
are separated by HPLC using a mobile phase of aqueous 0.1% (w/v)
L-cysteine.HCl.H2O + 0.1% (w/v) L-cysteine. Hg at mass-to-charge
ratio 202 vs. time is recorded and analyte peak areas are measured. Hg
species are identified by retention times of peaks. Methylmercury and
inorganic Hg concentrations are calculated using response factors
determined for standard solution prepared in aqueous 1% (w/v) L-
cysteine.HCl.H2O and peak areas measured in extracts. Total Hg is

47
 calculated as the sum of methyl and inorganic Hg concentrations
determined in extracts.
Apparatus/Instruments 1) Inductively coupled plasma-mass spectrometer—Capable of
measuring mass-to-charge ratio 202 in time resolved
(chromatographic) mode. Instrument should electronically interface
with or can be configured to remote start by standard HPLC
instruments for integrated operation.
2) High performance liquid chromatography—An integrated or
modular system consisting of an analytical pump and autosampler
capable of delivering aqueous mobile phase through analytical column
isocratically and programmed injection of acidic aqueous solutions.
3) HPLC analytical column—150 x 4.6 mm, 4 μm particle size.
4) Glass vials for extracting analytical samples—Amber, borosilicate
glass vials, 60 mL capacity, with screw caps.
5) Heated water bath—Capable of temperature control with sufficient
water and thermal capacity to allow immersion of extraction vials to
cap level and maintain water temperature at 60 ± 4 °C for 120
minutes.
6) Syringe for filtering extracts—Disposable, general use and non-
sterile.
7) Syringe filters for filtering extracts—Disposable, 0.45 μm
polypropylene membrane with polypropylene housing.
Materials and Reagents 1) Reagent water
2) Methylmercury(II) chloride—CH3HgCl crystals, purity ≥ 95%,
formula wt. 251.08.
3) Mercury(II) chloride—HgCl2 crystals, ACS grade, formula wt.
271.50.
4) L-cysteine hydrochloride monohydrate (L-cysteine.HCl.H2O)—Purity
> 98.5%, formula wt. 175.64.
5) L-cysteine (free base)—Purity ≥ 99.8%, formula wt. 121.16.
Preparation of Reagents 1) Extraction solution, aqueous 1% (w/v) L-cysteine.HCl.H2O—
Dissolve 10 ± 0.1 g L-cysteine.HCl.H2O crystals in 1000 ± 10 mL
reagent water.
2) Cysteine solution for preparation of standard solutions, aqueous 10%
(w/v) L-cysteine.HCl.H2O—Dissolve 5 ± 0.05 g L-cysteine.HCl.H2O
crystals in 50 ± 0.5 mL reagent water.
3) Mobil phase, aqueous 0.1% (w/v) L-cysteine + 0.1% (w/v) L-
cysteine.HCl.H2O—Dissolve 0.5 ± 0.01 g L-cysteine and 0.5 ± 0.01
g L-cysteine.HCl.H2O in 500 ± 5 mL reagent water.
4) Methylmercury stock solution, CH3HgCl in H2O that may
contain up to 20% (v/v) methanol, Hg=1000 mg/L—Tare 100-mL
volumetric flask on analytical balance in chemical fume hood. Weigh
0.1252 g CH3HgCl (FW=251.08) in flask with stopper in place. Add
≤ 20 mL methanol and swirl stoppered flask to dissolve CH3HgCl.
Dilute to 100.0 mL with reagent water. Discard solution in which

48
 inorganic Hg is > 3% of the theoretical methylmercury concentration.
5) Inorganic Hg stock solution, HgCl2 in 0.1% (v/v) HCl, Hg = 2000
mg/L—Tare 50-mL polypropylene centrifuge tube. Weigh 0.1354 g
HgCl2 (FW = 271.50) in tube. Add 5.0 ± 0.1 mL 1% (v/v) HCl and
swirl to dissolve. Dilute to 50.0 ± 0.5 mL with reagent water.
6) Multi-analyte intermediate solution, Hg due to CH3HgCl= 1000
μg/L and Hg due to HgCl2 = 1000μg/L in 0.02% (w/v) L
cysteine.HCl.H2O—Mix approximately 40mL reagent water and 0.1
mL 10% (w/v) L-cysteine.HCl.H2O in 50-mL polypropylene tube.
Add 50.0 μL methylmercury stock solution and 25.0 μL inorganic Hg
stock solution. Dilute to 50.0 ± 0.5 mL with reagent water.
7) Multi-analyte working standard solution, Hg due to CH3HgCl =1
μg/L and Hg due to HgCl2=1μg/L in 1% (w/v) L-
cysteine.HCl.H2O—Mix approximately 40 mL reagent water and 5.0
± 0.05 mL 10% (w/v) L-cysteine.HCl.H2O in 50-mL polypropylene
tube. Add 50.0μL multi-analyte intermediate solution. Dilute to 50.0 ±
0.5 mL with reagent water. Mix and immediately transfer a portion to
glass HPLC autosampler vial for storage before use.
8) Check solution—Use multi-analyte working standard solution for the
check solution.
9) Independent check solution (ICS)—Prepare independent inorganic
and methylmercury stock solutions, and independent multi-analyte
intermediate and working standard solutions according to steps (9) –
(12) from a different starting material than that used to prepare the
primary stock solutions.
Sample Preparation 1) Weigh analytical portion into 60-mL amber glass extraction vial and
determine mass of analytical portion. Generally, weigh 0.5 ± 0.1 g
edible portion of seafood. Use 0.2 ± 0.01 g for reference materials.
2) Add 50.0 ± 0.5 mL extraction solution (aqueous 1% (w/v) L-
cysteine.HCl.H2O) to extraction vials, cap tightly, and shake
vigorously by hand.
3) Heat extraction vials 120 ± 5 min in water bath at 60 ± 4 °C. Shake
each vial vigorously by hand after 60 minutes of heating and again
after 120 minutes of heating.
4) Remove extraction vials from water bath and allow cooling to room
temperature.
5) Filter a portion of extract through 0.45 μm filter directly into HPLC
autosampler vial.
Method of analysis The optimum operating settings and conditions must be determined for
the equipment used.
Instrument Setup
1) Setup and configure HPLC and ICP-MS separately before connecting
equipment together. Follow instrument standard operating procedures
for startup and initialization.
• Tune ICP-MS normally. Ensure instrument performance meets

49
 default specifications for sensitivity, precision, stability, and/or other
established system suitability requirements.
• Set ICP-MS data acquisition for mass-to-charge ratio 202 in time
resolved mode with 1 replicate (read) per point and use an initial
dwell (integration) time of 1 second per point.
• Purge and condition HPLC and analytical column with mobile
phase.

2) Connect HPLC to ICP-MS.

• Enable communication between instruments to synchronize ICP-MS
data acquisition with HPLC injection start.
• Stop HPLC flow and connect column output directly to ICP
nebulizer using tubing and fittings.

3) Optimize operating conditions.

• Start HPLC flow and ensure proper liquid flow through ICP
nebulizer and drainage of spray chamber.
• Analyze a multi-analyte standard solution and adjust acquisition
parameters to obtain 10-20 data points across narrowest analyte
peak.
• Monitor instrument conditions to ensure operation is stable and
within normal functioning range.

4) Check instrument performance.

• Verify baseline resolution between inorganic and methylmercury
peaks and that peaks are not tailing excessively.
• Analyze a multi-analyte standard solution 3 or more times and
verify short term precision is less than 5% relative standard
deviation (peak area) for all analyte(s) of interest.
• Verify absence of instrument carry-over.

Determination of Analyte Concentration Using Response Factor

1) Analyze a multi-analyte standard solution (or single analyte standard
solutions separately) and extraction solution 2 or more times each.
2) Calculate response factors and check accuracy of working standard(s).
• Analyze independent check solution(s). Acceptance criteria:
recovery within 100 ± 5%.
3) Analyze analytical solutions and quality control solutions.
4) Check instrument measurement performance
• Check solution analyzed at a frequency of 10% and at end of the
analytical run has a recovery of 100 ± 10% (continuing calibration
verification).
• Extraction solution analyzed following each check solution
analysis is < ASDL (verify absence of carry-over).
• Measurements do not surpass the LDR. Dilute analytical solution

50
 with extraction solution if necessary to comply with criteria.
Retention time of analyte peaks of analytical solution is
comparable to standard solution.
Calculation with units of Calculate response factor of analyte, RF (cps-s/μg/L)
expression
RF = Astd-ave- Aes-aveCstd
Cstd
where
Astd-ave= average peak area of n > 2 injections of standard
solution(s) (cps-s)
Aes-ave= average peak area of n > 2 injections of extraction solution
(cps-s) (0 if no peak is detected)
Cstd= analyte concentration (μg/L) in standard solution(s)

Calculate concentration of analyte (inorganic mercury or methylmercury)

in analytical solution, S (μg/L)
S = Aas −Aes−ave
RF
where
Aas= peak area of analyte in analytical solution (cps-s)
Aes-ave= average peak area of analyte in extraction solution (cps-s)
(0 if no peak is detected)
RF = response factor of analyte (cps-s / μg/L)

Calculate concentration of total Hg in analytical solution, ST (μg/L)

ST = Sinorg+Smethyl
where
Sinorg= concentration of inorganic Hg in analytical solution (μg/L)
Smethyl= concentration of methyl Hg in analytical solution (μg/L)

Calculate the concentration (mass fraction) of analyte in the

analytical portion according to the formula
Concentration (μg/kg)= [(ST×DF)−MBKL]×V
m×MCF
where
ST = concentration of analyte (S or total Hg, ST) in analytical
solution (or diluted analytical solution) (μg/L)
MBKL = laboratory MBK (μg/L)
V = volume (L) of analytical solution (0.050 L)
m = mass of analytical portion (kg)
DF = dilution factor (1 if analytical solution not diluted)
MCF = mass correction factor (1 if water or other solvent not added
to aid homogenization)
Inference

51
(Qualitative Analysis)
Reference U.S. Food and Drug Administration-(4.8) High Pressure Liquid
Chromatographic-Inductively Coupled Plasma-Mass Spectrometric
Determination of Methylmercury and Total Mercury in Seafood (version
1.0)
Approved by Scientific Panel on Methods of Sampling and Analysis

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