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  5. Rapid Determination of Perchlorate Anion in Foods by Ion Chromatography-Tandem Mass Spectrometry
  1. Environmental Contaminants in Food

Rapid Determination of Perchlorate Anion in Foods by Ion Chromatography-Tandem Mass Spectrometry

Revision 2: April 12, 2005

Purpose

To provide an analytical method for the quantitative determination of perchlorate anion in fruits, vegetables, milk, low moisture foods, infant foods, and water.

Revision Information

This section will identify differences between the present method and the last method (Revision 1: July 27, 2004) posted on the website. This is the second revision of the method.

Summary of changes in the current revision are as follows:

Additional Commodities: Method now includes low moisture foods such as oats, wheat flour, and corn meal.

Extraction Solution: 1% acetic acid in water is used to extract all commodities except milk. For milk, infant foods, and low moisture foods, 1% acetic acid solution in water is used in conjunction with acetonitrile. The purpose of 1% acetic acid solution is to prevent retention of perchlorate on graphitized carbon SPE. The acetonitrile in extraction solution makes it less cloudy.

Apparatus: Syringe filters used: Syringe Filters: PTFE; 0.20 µm pore size × 25 mm; P/N 2089; Alltech; Deerfield, IL. Nylon syringe filters tend to retain perchlorate.

Bottled Water: Bottled water need not be filtered, prior to IC-MS/MS.

Mass Spectrometry Conditions: For those using Waters Micromass equipment, we have found that lowering the capillary voltage from 3.25 kV to 0.58 kV improves sensitivity by two fold. However, this parameter should be determined for each instrument used to analyze perchlorate.

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, and 3.0 ppb in low moisture foods such as oats, wheat flour, corn meal, and cattle feed.

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.

  1. Analytical Standard native perchlorate): Perchlorate (ClO4-); 997±6 µg/mL; Alltech; Deerfield, IL; Catalog No. 37048.
  2. Internal Standard: 18O4-labeled sodium perchlorate; 98+%; Icon Services Inc., Mt. Marion, NY; Catalog No. IO 6455sp.
  3. Acetonitrile: HPLC Grade; "Baker Analyzed"; J.T. Baker; Phillipsburg, NJ.
  4. 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.
  5. Ammonium Acetate: HPLC Grade; 98.7% purity; Fisher Scientific; Fair Lawn, NJ.
  6. Glacial Acetic Acid: ACS Reagent; Fisher Scientific; Fair Lawn, NJ.
  7. Extraction solution (1% acetic acid in water): Add 10 mL glacial acetic acid to 900 deionized water and mix.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. 0.25 µg/L native ClO4- + 10 µg/L IS.
  13. 0.50 µg/L native ClO4- + 10 µg/L IS.
  14. 1.0 µg/L native ClO4- + 10 µg/L IS.
  15. 2.0 µg/L lO4- + 10 µg/L IS.
  16. 5.0 µg/L native ClO4- + 10 µg/L IS.
  17. 10.0 µg/L native ClO4- + 10 µg/L IS.
  18. 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.

  1. General Laboratory Glassware and Containers: Assorted beakers, volumetric flasks, volumetric pipets, and 4 oz. storage bottles with Teflon-capped liners.
  2. Microliter Syringe: Hamilton; 100 µL capacity; Hamilton Company; Reno, NV.
  3. Syringe Filters: PTFE; 0.20 µm pore size × 25 mm; P/N 2089; Alltech; Deerfield, IL..
  4. Disposable Syringes: 3 mL syringe (for syringe filters); Beston Dickinson and Co.; Franklin Lakes, NJ.
  5. HPLC Solvent Filters: 0.20 µm pore size × 47 mm Millipore; Millipore Corp., Bedford, MA.
  6. Concentration Tubes: 1.0 mL graduated concentration tubes; Catalog No. 570005-0124; Kontes.
  7. 50 mL polypropylene conical tubes: Blue Max™, 50 mL; Catalog No. 352098. Becton Dickinson Labware; Franklin Lakes, NJ.
  8. Supelclean™, Envi™-Carb SPE cartridges, 500 mg bed × 6 mL; P/N 57094; Supelco, Bellefonte, PA.
  9. Supelco Visiprep 24™ SPE manifold; Supelco, Bellefonte, PA.
  10. Centrifuge equipped with 50 mL holders: IEC HN-SII; Needham Heights, MA.
  11. HPLC: Agilent Model 1100 autosampler; binary HPLC pump and column heater (Palo Alto, CA).
  12. IC Column: Waters IC-Pak™ Anion HR; 4.6 mm × 75 mm; P/N WAT026765 (Milford, MA).
  13. Guard Column: Waters IC-Pak™ Anion Guard-Pak™ Kit (Guard-Pak™ Holder and five inserts); P/N WAT007357 (Milford, MA).
  14. Mass Spectrometer: Micromass Quattro Micro™ triple stage quadrupole mass spectrometer (Manchester, UK).
  15. Mass Spectrometry Software: Micromass Masslynx™ version 3.5 Manchester, UK).

Sample Preparation for High Moisture Fruits and Vegetables

  1. Chop commodity with food processor or equivalent device, until matrix appears consistent. For cattle feed, it is recommended to chop while frozen.
  2. Weigh 10 g test portion of chopped commodity into a 50 mL polypropylene conical tube. For cattle feed, weigh a 5.0 g test portion of chopped commodity into a 50 mL polypropylene conical tube.
  3. 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)
  4. Add 20 mL 1% acetic acid in water, cap tube, and shake by hand for 2 min. For cattle feed, add 30 mL 1% acetic acid in water, cap tube, nd shake by hand for 2 min
  5. Centrifuge tube containing test portion 15 min at 2,000 rpm.
  6. 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.
  7. 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.
  8. Filter sample extract with 0.20 µm pore size nylon syringe filter, prior to IC-MS/MS analysis.
  9. Assume that the total volume of sample extract is 30 mL. The internal standard will correct for any error.

Sample Preparation for Bottled Water

  1. 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.
  2. Carefully add 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. Bottled water need not be filtered with 0.20 µm PTFE syringe filter.
  3. 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

  1. Pipette 5.0 mL, (using a volumetric pipett) of test portion into 50 mL polypropylene conical tube.
  2. 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 1% acetic acid in water and 20 mL acetonitrile. Cap centrifuge tube and briefly shake solution by hand or vortex solution.
  3. Centrifuge test portions at 2,000 rpm for five minutes.
  4. Condition Supelclean™ Envi™ Carb SPE cartridge with 6 mL acetonitrile, followed by 6 mL 1% acetic acid in water. Discard acetonitrile and water portions used to prepare cartridges.
  5. 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.
  6. 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)
  7. Filter sample with 0.20 µm pore size nylon syringe filter, prior to IC-MS/MS analysis.

Sample Preparation for Low Moisture Foods (Oats, Corn Meal, and Wheat Flour)

  1. Grind commodity with food processor or equivalent device, until matrix appears consistent. (This is not necessary with the fine granular or powdery commodities such as wheat flour and corn meal).
  2. Weigh 5.0 g test portion of ground commodity into a 50 mL polypropylene conical tube.
  3. Using a 100 µL syringe, add 100 µL of 4.0 µg/mL (400 ng) labeled internal standard to test portion. (Assuming a total volume of sample extract is 40 mL, the final concentration of internal standard will be 10 µg/L)
  4. Add 20 mL 1% acetic acid in water together with 20 mL acetonitrile, cap tube, and shake by hand for 2 min. Purpose of the acetonitrile is to prevent clumps from forming, particularly with wheat flour.
  5. Centrifuge tube containing test portion 15 min at 2,000 rpm.
  6. Condition Supelclean™ Envi™ Carb SPE cartridge with 6 mL acetonitrile, followed by 6 mL 1% acetic acid in water and allow to dry (with vacuum) for 30 sec. Discard aqueous portion used to prepare cartridges.
  7. 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.
  8. Filter sample extract with 0.20 µm pore size PTFE syringe filter, prior to IC-MS/MS analysis.
  9. Assume that the total volume of sample extract is 40 mL. The internal standard will correct for any error.

Sample Preparation for Infant Foods

  1. Weigh 10 g test portion of homogenized infant food into a 50 mL polypropylene conical tube.
  2. 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)
  3. Add 15 mL of acetonitrile, cap 50 mL centrifuge tube and briefly shake by hand for 1 min. Add 5 mL 1% acetic acid in water, cap tube, and shake by hand for 2 min.
  4. Centrifuge tube containing test portion 15 min at 2,000 rpm.
  5. Condition Supelclean™ Envi™ Carb SPE cartridge with 6 mL acetonitrile, followed by 6 mL 1% acetic acid in water and allow to dry (with vacuum) for 30 sec. Discard aqueous portion used to prepare cartridges.
  6. 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.
  7. Filter sample extract with 0.20 µm pore size PTFE syringe filter, prior to IC-MS/MS analysis.
  8. Assume that the total volume of sample extract is 30 mL. The internal standard will correct for any error.

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.

  1. HPLC Mobile Phase Composition: 100 mM ammonium acetate in 50:50 (v/v) water/acetonitrile
  2. IC Column: 4.6 mm × 75 mm Waters IC-Pak™ Anion HR
  3. Guard Column: Waters IC-Pak™ Anion Guard-Pak™
  4. Column Flow: 300 µL/min, isocratic
  5. Injection Volume: 50 µL
  6. Column Temperature: 35°C
  7. Perchlorate Anion Elution Time: Approximately 11.0 min
  8. Ionization Mode: Negative Ion Electrospray
  9. Capillary Voltage: 0.58 kV
  10. Cone Voltage: 50 V
  11. Extractor Voltage: 1.0 V
  12. RF Lens Voltage: 0.0 V
  13. Source Temperature: 120°C
  14. Desolvation Temperature: 350°C
  15. Cone Gas Flow: 60 L/h nitrogen
  16. Desolvation Gas Flow: 510 L/h nitrogen
  17. Collision Gas Pressure: 2 × 10-3 mbar Argon
  18. Collision Energy: 30 eV
  19. Multiple Reaction Monitoring (MRM) Ions:
    1. m/z 99→83, primary transition for native perchlorate (quantitation)
    2. m/z 101→85, secondary transition for native perchlorate (confirmatory)
    3. m/z 107→89, primary transition for labeled IS (quantitation).
    4. m/z 109→91, secondary transition for labeled IS (confirmatory).
    5. Dwell time of 0.5 sec with 0.10 seconds inter channel delay.
    6. Peak areas are used for quantitation.
  20. 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.
  21. 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.
  22. 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.
  23. 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 follows:

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) × (ISSTD ÷ ISSMP)

For fruits and vegetables: ppb = (C × TV) ÷ W

For Bottled Water: µg /L = C × (FV in Concentrator Tube ÷ VS)

For Milk: µg /L = ((C × TV) ÷ V)

% Recovery = (ppb Found ÷ ppb added) × 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) × (ISSTD ÷ ISSMP)
C = ((0.046003 - 0.003826) ÷ 0.07067) × (10 ÷ 10) = 0.60 µg/L

ppb = (C × TV) ÷ W
ppb = (0.60 × 30) ÷ 10.00 = 1.80 ppb found

% Recovery = (ppb found ÷ ppb fortified) × 100
% Recovery = (1.80 ÷ 1.99) × 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: Alex.Krynitsky@fda.hhs.gov

Press inquiries MUST be directed to:

FDA Press Office
Shelly Burgess, Press Officer
Phone: 301-796-4651
Email: Shelly.Burgess@fda.hhs.gov
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