DRAFT
Rapid Determination of Perchlorate Anion in Foods by Ion Chromatography-Tandem Mass
Spectrometry
Revision 1
Purpose
To
provide an analytical method for the quantitative determination of perchlorate
anion in fruits, vegetables, milk, and water.
Revision Information
This section will identify differences between the present method
and the last method (Revision 0: March 17, 2004) posted on the website.
This is the first revision of
the method.
Summary of changes in the current revision are as follows:
Title: Title
has been renamed to: "Rapid Determination of Perchlorate Anion in Foods by Ion
Chromatography-Tandem Mass Spectrometry". Note: No changes were made with HPLC
conditions or the mass spectrometry conditions.
Reagents: Nitric
acid is not needed.
Apparatus:
Syringe filters used: Nylon; 0.20 µm pore
size x 30 mm; P/N 42225-NN; Sun SRI Titan; Eatontown, NJ. The following are no longer needed: Glass fiber filters, vacuum filter
adapters, Buchner funnels, stoppered graduated cylinders, and conical-bottom
glass centrifuge tubes. Conical-bottom centrifuge tubes are replaced with
disposable 50 mL polypropylene conical tubes.
Sample
Preparation for Lettuce: Sample
preparation procedure for lettuce has been renamed to "Sample Preparation
for Fruits and Vegetables". Applicability of commodities include: lettuce,
cantaloupe, carrots, spinach, citrus, and tomatoes. The current procedure is less
time consuming than the previous procedure for lettuce (Revision 0: March 17, 2004). The
modified procedure is as follows: A 10 g test portion of chopped fruit or
vegetable is weighed into a 50 mL polypropylene conical tube, followed by the
addition of the isotopically labeled internal standard. 20 mL of deionized
water is added and the 50 mL tube is capped and shaken by hand for 2 min.
Sample tube is then centrifuged at 2,000 rpm for 15 min and a 5 mL portion of
supernatant is passed through a graphitized carbon solid phase extraction (SPE)
cartridge. The eluant is collected and filtered through a 0.20 µm nylon syringe
filter, prior to ion
chromatography-tandem mass spectrometry (IC-MS/MS) determination. Please see current procedure for complete details.
Principle
Individual
procedures for foods involve extraction, graphitized carbon solid phase
extraction (SPE) cleanup, filtration, followed by ion chromatography-tandem
mass spectrometry (IC-MS/MS) determination. A triple stage quadrupole mass
spectrometer, equipped with electrospray ionization (ESI) in the negative mode,
is used to determine perchlorate anion. An 18O4-labeled perchlorate
internal standard is used to correct for any matrix effects. The limit of quantitation
(LOQ) is 1.0 ppb in fruits and vegetables, 0.50 µg/L in bottled water, and 3.0
µg/L in milk.
Safety Precautions
Restrict
exposure to all chemicals used in this method. Use safety gloves,
glasses/goggles, laboratory coat, ventilation, and safe handling techniques. Review
appropriate Material Safety Data Sheets (MSDS) for all chemicals used prior to
starting work.
Reagents
Items
from manufacturers other than those listed may be used if they are shown to be
functionally equivalent.
- Analytical Standard
(native perchlorate): Perchlorate (ClO4-); 997±6 µg/mL;
Alltech; Deerfield, IL; Catalog No. 37048.
- Internal Standard: 18O4-labeled
sodium perchlorate; 98+%; Icon Services Inc., Mt. Marion, NY; Catalog
No. IO 6455sp.
- Acetonitrile: HPLC Grade; "Baker
Analyzed"; J.T. Baker; Phillipsburg, NJ.
- Deionized (DI)
Water: Pass water through a Millipore Milli-Q-Pure Ultra-Pure water filtration
system. Use this
water for all solutions and steps requiring water.
- Ammonium Acetate:
HPLC Grade; 98.7% purity; Fisher Scientific; Fair Lawn, NJ.
- HPLC Mobile Phase:
100 mM ammonium acetate in 50:50 (v/v) acetonitrile: water. Weigh 7.71 grams
ammonium acetate and dissolve with 500 mL
deionized water. After ammonium acetate has dissolved, 500 mL of acetonitrile
is then mixed with aqueous solution. The final mobile phase solution is then
filtered through a 47 mm, 0.20 µm Millipore solvent filter prior
to HPLC use.
- Internal Standard
(IS) Solutions: Accurately weigh 0.0121 g of 18O4-labeled
sodium perchlorate and dilute with DI water in 100 mL volumetric flask.
This is equivalent to 100 µg/mL 18O4-labeled ClO4-.
From the 100 ug/mL stock solutions, prepare the following working standards:
10 µg/mL,
4.0 µg/mL,
3.0 µg/mL,
1.0 µg/mL,
and 0.10 µg/mL. Solutions are prepared in 100 mL volumetric flasks with DI water
and stored in 4 oz bottles with Teflon-capped liners. Standards are stored in
a refrigerator at 4°C.
- Native Perchlorate Standard
Solutions (used for fortification and subsequent preparation of IC-MS/MS
working standards): Starting with the native perchlorate analytical standard
(997±6 µg/mL), prepare the following working standards: 100 µg/mL, 10 µg/mL, 1.0 µg/mL, 0.10 µg/mL, and 0.010
µg/mL.
Solutions are prepared in 100 mL volumetric flasks with DI water and stored in 4
oz. bottles with Teflon-capped liners. Standards are stored in a refrigerator
at 4°C.
- IC-MS/MS working standards:
Starting with the native perchlorate standards (item 9) and 1.0 µg/mL 18O4-labeled ClO4- working
standard, the following solutions are made for IC-MS/MS quantitation.
The concentration of the internal standard (IS) is kept constant at 10 µg/L (made
by adding 1.0 mL of 1.0 µg/mL labeled IS in 100 mL of solution). Note that the
values herein are expressed in µg/L and Not µg/mL. Solutions are
prepared in 100 mL volumetric flasks with DI water and stored in 4 oz bottles
with Teflon-capped liners. Standards are stored in a refrigerator at 4°C.
- 0.25 µg/L native ClO4- + 10 µg/L IS.
- 0.50 µg/L native ClO4- + 10 µg/L IS.
- 1.0 µg/L native ClO4- + 10 µg/L IS.
- 2.0 µg/L ClO4- + 10 µg/L IS.
- 5.0 µg/L native ClO4- + 10 µg/L IS.
- 10.0 µg/L native ClO4- + 10 µg/L IS.
- 100 µg/L native ClO4- + 10 µg/L IS.
Apparatus
Items
from manufacturers other than those listed may be used if they are shown to be
functionally equivalent.
- General Laboratory Glassware and Containers: Assorted beakers, volumetric flasks,
volumetric pipets, and 4 oz. storage bottles with Teflon-capped liners.
- Microliter Syringe: Hamilton; 100 µL capacity; Hamilton Company; Reno,
NV.
- Syringe Filters: Nylon; 0.20 µm pore size x 30 mm; P/N 42225-NN;
Sun SRI Titan; Eatontown, NJ.
- Disposable Syringes: 3 mL syringe (for syringe filters); Beston Dickinson
and Co.; Franklin Lakes,
NJ.
- HPLC Solvent Filters: 0.20 µm pore size x 47 mm Millipore; Millipore
Corp., Bedford, MA.
- Concentration Tubes: 1.0 mL graduated concentration tubes; Catalog No. 570005-0124; Kontes.
- 50 mL polypropylene conical tubes: Blue Max™,
50 mL; Catalog No. 352098. Becton Dickinson Labware; Franklin Lakes,
NJ.
- Supelclean™, Envi™-Carb
SPE cartridges, 500 mg bed x 6 mL; P/N 57094; Supelco, Bellefonte,
PA.
- Supelco Visiprep 24™ SPE manifold; Supelco, Bellefonte, PA.
- Centrifuge equipped with 50 mL holders: IEC HN-SII; Needham Heights, MA.
- HPLC: Agilent Model 1100 autosampler; binary HPLC pump and column
heater (Palo Alto,
CA).
- IC Column: Waters IC-Pak™ Anion HR; 4.6 mm x 75 mm; P/N WAT026765
(Milford, MA).
- Guard Column: Waters IC-Pak™ Anion Guard-Pak™ Kit (Guard-Pak™ Holder
and five inserts); P/N WAT007357 (Milford, MA).
- Mass Spectrometer: Micromass Quattro Micro™ triple stage
quadrupole mass spectrometer (Manchester, UK).
- Mass Spectrometry Software: Micromass Masslynx™ version 3.5
(Manchester, UK).
Sample Preparation for Fruits
and Vegetables
- Chop commodity with food
processor or equivalent device, until matrix appears consistent.
- Weigh 10 g test portion of
chopped commodity into a 50 mL polypropylene conical tube.
- Using a 100 µL syringe, add 100 µL
of 3.0 µg/mL (300 ng) labeled internal standard to test portion. (Assuming a
total volume of sample extract is 30 mL, the final concentration of internal
standard will be 10 µg/L)
- Add 20 mL deionized water,
cap tube, and shake by hand for 2 min.
- Centrifuge tube containing
test portion 15 min at 2,000 rpm.
- Condition Supelclean™ Envi™ Carb
SPE cartridge with 6 mL water and allow to dry (with vacuum) for 30
sec. Discard water portion used to prepare cartridges.
- Load Supelclean™ Envi™ Carb
SPE cartridge with approximately 5 mL supernatant (one column volume)
and collect eluant in 50 mL polypropylene conical tube. When decanting
supernatant
into SPE, some particulates may enter SPE. This is acceptable as
long as particulates do not clog SPE. Adjust vacuum in Visiprep so
that the SPE flow
is approximately two drops per second.
- Filter sample extract with 0.20 µm
pore size nylon syringe filter, prior to IC-MS/MS analysis.
- Assume that the total volume
of sample extract is 30 mL. The internal standard will correct for
any error.
Sample Preparation for Bottled
Water
- Using a 100 µL syringe, add 100 µL
of 0.10 µg/mL (10 ng) labeled internal standard to a 1.0 mL concentration
tube.
- Using a 3 mL disposable syringe
and a 0.45 µm pore size nylon syringe filter, filter test portion of bottled
water directly into concentration tube until the final volume is exactly 1.0 mL.
Hence the amount of IS is 0.10 mL and volume of bottled water test portion is
0.90 mL. The concentration of IS in final solution is 10 µg/L.
- Shake final solution by hand
or briefly vortex to disperse test potion and labeled IS in solution,
prior to IC-MS/MS
analysis.
Sample Preparation for Milk
- Pipette
5.0 mL, (using a volumetric pipett) of test portion into 50 mL polypropylene
conical tube.
- Using
a 100 µL syringe, add 100 µL of 4.0 µg/mL
(400 ng) labeled internal standard to test portion and briefly
swirl centrifuge tube. Add 5.0 mL deionized
water and 20 mL acetonitrile. Cap centrifuge tube and briefly
shake solution by hand or vortex solution.
- Centrifuge
test portions at 2,000 rpm for five minutes.
- Condition
Supelclean™ Envi™ Carb SPE cartridge
with 6 mL acetonitrile, followed by 6 mL water. Discard
acetonitrile and water portions used to
prepare cartridges.
- Load
Supelclean™ Envi™ Carb SPE cartridge
with supernatant and collect eluant in 50 mL polypropylene
conical tube.
Adjust vacuum in Visiprep so that the SPE flow is approximately
two drops per second. After supernatant has completely
passed through, wash SPE
with 6 mL of deionized water and collect eluant in the
above collection vessel.
- Adjust
final volume of sample extract to 40 mL with deionized
water and briefly vortex centrifuge tube. (Assuming a total volume
of sample
extract is 40 mL, the
final concentration of internal standard will be 10 µg/L)
- Filter
sample with 0.20 µm pore size nylon syringe filter,
prior to IC-MS/MS analysis.
IC-MS/MS Conditions and Setup
1.0 µg/mL
standards (diluted in HPLC mobile phase) for each compound, native perchlorate
and isotopically labeled IS, are tuned and the parameters that give the optimal collision energy are determined for each
transition recorded by multiple reaction monitoring (MRM). Our best collision
energy settings, desolvation gas flow, and cone gas flow are reported. The
settings listed have been determined for the Micromass Quattro Micro™ triple
stage quadrupole mass spectrometer. These parameters should be determined for
each instrument used to analyze perchlorate.
- HPLC Mobile Phase
Composition: 100 mM ammonium acetate in 50:50 (v/v) water/acetonitrile
- IC Column: 4.6 mm x 75 mm
Waters IC-Pak™ Anion HR
- Guard Column: Waters IC-Pak™ Anion
Guard-Pak™
- Column Flow: 300 µL/min, isocratic
- Injection Volume: 50 µL
- Column Temperature: 35°C
- Perchlorate Anion Elution
Time: Approximately 11.0 min
- Ionization Mode: Negative
Ion Electrospray
- Capillary Voltage: 3.25 kV
- Cone Voltage: 50 V
- Extractor Voltage: 1.0 V
- RF Lens Voltage: 0.0 V
- Source Temperature: 120°C
- Desolvation Temperature: 350°C
- Cone Gas Flow: 60 L/h nitrogen
- Desolvation Gas Flow: 510 L/h
nitrogen
- Collision Gas Pressure: 2 x 10-3 mbar
Argon
- Collision Energy: 30 eV
- Multiple Reaction Monitoring (MRM)
Ions:
- m/z 99→83, primary
transition for native perchlorate (quantitation)
- m/z 101→85, secondary
transition for native perchlorate (confirmatory)
- m/z 107→89, primary
transition for labeled IS (quantitation).
- m/z 109→91, secondary
transition for labeled IS (confirmatory).
- Dwell time of 0.5 sec
with 0.10 seconds inter channel delay.
- Peak areas are used
for quantitation.
- The analytical solution quantitation
limit (ASQL) is defined as the level at which a 10:1 peak to peak signal/noise
ratio is observed for the analyte quantitation ion transition (m/z 99→83).
The limit of quantitation (LOQ) is the ASQL adjusted for the analytical
portion’s mass and dilution.
- The analytical solution detection
limit (ASDL) is defined as the level at which a 3:1 peak to peak signal/noise
ratio is observed for the analyte quantitation ion transition (m/z 99→83).
The limit of detection (LOD) is the analytical solution detection limit adjusted
for the analytical portion’s mass and dilution.
- Confirmation: Retention time of sample
peak must match the average retention time of perchlorate standards, in
calibration curve (± 0.15 minute). Also the ratio of the following MRM
ion transitions: peak area m/z 101→85 ÷ peak area m/z 99→83, must
match within ± 10 % of the average values of the standards, in
calibration curve. For perchlorate the average ratio, peak are m/z 101→85
÷ peak area m/z 99→83, is ~ 0.35. This value may vary, depending
on how the instrument is tuned.
- Calibration curve and sample
sequence: This method was developed using
a seven point calibration curve, with the following standard solutions: 0.25 µg/L,
0.50 µg/L, 1.0 µg/L, 2.0 µg/L, 5.0 µg/L, 10.0 µg/L, and 100 µg/L. The
correlation coefficient should be at least 0.999. A sample set consists of: standards
used in calibration curve, twenty samples, one control (of the matrix analyzed),
one method reagent blank, one standard check at 1.0 µg/L, and one standard
check at 5.0 µg/L. The standard checks should not differ more than ± 10%
from actual values.
Calculations
A
formula for calculation of perchlorate in ppb or µg/L (in the case of milk and
bottled water) is provided.
For
each data point a ratio is calculated as followed:
Resp.
Fact. = peak area of m/z 99→83 ÷ peak area of m/z 107→89
Concentration
in µg/L is calculated as follows using data generated from the calibration
curve:
C = ((Resp.
Fact. of sample – b) ÷ m) x (ISSTD ÷ ISSMP)
For fruits
and vegetables: ppb = (C x TV) ÷ W
For
Bottled Water: µg /L = C x (FV in Concentrator Tube ÷ VS)
For
Milk: µg /L = ((C x TV) ÷ V)
%
Recovery = (ppb Found ÷ ppb added) x 100. This calculation is used when
fortifying control matrices in order to check performance of method.
Where:
- Resp.
Fact. = Response Factor
- C =
Concentration in µg/L
- b = Y
intercept from calibration curve
- m =
Slope from calibration curve.
- ISSTTD
= Concentration of internal standard in native standard (10 µg/L
as outlined in method).
- ISSMP =
Concentration of internal standard in sample extract (10 µg/L
as outline in method).
- TV =
Total volume of extract (30 mL for fruits and vegetables and 40 mL
for milk).
- W =
Weight of test portion.
- V =
Volume of test portion (for milk and bottled water).
- FV =
Final volume of sample extract + IS in Concentrator tube (1.0 mL).
- VS =
Volume of sample extract in concentrator tube (0.90 mL).
Example
of Calculation (Lettuce Sample):
peak area of m/z 99→83 = 62.90
peak area of m/z 107→89 = 1367.31
b = 0.003826
m = 0.07067
ISSTTD = 10 µg/L
ISSMP = 10 µg/L
TV = 30 mL
W = 10.00 g
ppb fortified = 1.99 ppb
RF = peak area m/z 99→83 ÷ peak area m/z 107→89
RF = 56.74 ÷ 1367.31 = 0.046003
C = ((RF of sample – b) ÷ m) x (ISSTD ÷ ISSMP)
C = ((0.046003 – 0.003826) ÷ 0.07067) x (10 ÷ 10) = 0.60 µg/L
ppb = (C x TV) ÷ W
ppb = (0.60 x 30) ÷ 10.00 = 1.80 ppb found
% Recovery = (ppb found ÷ ppb fortified) x 100
% Recovery = (1.80 ÷ 1.99) x 100 = 90.5 %
Comments or questions on method may be directed to:
Alexander J. Krynitsky
Chief, Methods Research Branch
FDA/CFSAN/DPIC/MRB/HFS-336
5100 Paint Branch Parkway
College Park, MD 20740-3835
Phone: 301-436-2098
Email:
Press inquiries MUST be directed to:
FDA Press Office
Kimberly Rawlings
Press Officer
301-436-2288