Attachment 2.pdf

Overview of Method for Analysis of Nitrite and Nitrate in Pig Liver Using Liquid Chromatography with Ultraviolet Radiation Detection

Jon C. Andre (Battelle) Page 1 of 6 02October2020

1. Equipment and Chemicals (Equivalent Substitutions Possible)

Identifier Manufacturer Model No. or Catalog No.

Analytical Balance Mettler-Toledo

Weight Set Troemer

Positive Displacement Pipet Gilson M1000

Positive Displacement Pipet Gilson M250

Positive Displacement Pipet Gilson M25

Centrifuge Beckman Coulter J2-HS

Centrifuge Beckman Coulter GS-6R

Macrosep Centrifugal Concentrator

Pall Corporation (supplier VWR) 89131-980

Sample Rotator

Refrigerator

Freezer

Sodium Nitrite Fisher Scientific S347

Sodium Nitrate Fisher Scientific S343

Sodium Hydroxide (50% w/w) Fisher Scientific SS410-4

Pig Liver BioIVT

ASTM Type 1 Water Aqua Solutions

2. Preparation of Solutions

2.1 Mobile Phase A: 1 mM Sodium Hydroxide Solution

Transfer 80 µL of 50% sodium hydroxide solution to a 2-L volumetric flask. Adjust the volume of the flask by the addition of ASTM Type 1 water. Mix thoroughly the solution and store it at room temperature.

2.2 Mobile Phase B: 100 mM Sodium Hydroxide Solution

Transfer 8 mL of 50% sodium hydroxide solution to a 2-L volumetric flask. Adjust the volume of the flask by the addition of ASTM Type 1 water. Mix thoroughly the solution and store it at room temperature.

Attachment 2, 12639521Q0024

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2.3 0.5 M Sodium Hydroxide Solution

Transfer 20 mL of 50% sodium hydroxide solution to a 1-L volumetric flask. Adjust the volume of the flask by the addition of ASTM Type 1 water. Mix thoroughly the solution and store it at room temperature.

2.4 10 mM Sodium Hydroxide Solution

Transfer 40 mL of 0.5 M sodium hydroxide solution to a 1-L volumetric flask. Adjust the volume of the flask by the addition of ASTM Type 1 water. Mix thoroughly the solution and store it at room temperature.

2.5 Preparation of Analytical Standard Solutions

2.5.1 Preparation of Nitrate Stock Standard Solutions

Weigh the amounts of sodium nitrate indicated in Table 1 into individual volumetric flasks. Dilute to volume with ASTM Type I water. Mix thoroughly the solutions and store them at room temperature.

Table 1. Preparation of Nitrate Stock Standard Solutions aNitrate in sodium nitrate (62.005/84.995 g/mol)

2.5.2 Preparation of Nitrite Stock Standard Solutions

Weigh the amounts of sodium nitrite indicated in Table 2 into individual volumetric flasks. Dilute to volume with ASTM Type I water. Mix thoroughly the solutions and store them at room temperature.

Table 2. Preparation of Nitrite Stock Standard Solutions aNitrite in sodium nitrate (46.005/68.995 g/mol)

2.5.3 Preparation of Standard Spiking Solutions (SS)

Using a positive displacement pipet, transfer the volume of each source solution indicated in Table 3 into individual volumetric flasks. Dilute to volume with 10 mM sodium hydroxide solution. Mix thoroughly the solutions and store them at room temperature.

Solution ID Target

Concentration (µg/mL)a

Target Weight

(mg) Actual Weight (mg) Final Volume

(mL)

Nitrate A 1,000 34.4 ± 2 25

Nitrate B 1,000 34.4 ± 2 25

Nitrate C 2,000 68.8 ± 2 25

Solution ID Target

Concentration (µg/mL)a

Target Weight

(mg) Actual Weight (mg) Final Volume

(mL)

Nitrite A 1,000 38.6 ±2 25

Nitrite B 1,000 38.6 ± 2 25

Nitrite C 2,000 77.2 ± 2 25

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Table 3. Preparation of Standard Spiking Solutions (SS)

Solution ID Nitrate Target Concentration

(µg/mL)

Nitrite Target Concentration

(µg/mL)

Source Solution

Source Volume (mL)

Final Volume (mL)

SS1 400 400

Nitrate A 4

Nitrite A 4

SS2 200 200

Nitrate B 2

Nitrite B 2

SS3 40 40 SS1 1 10

SS4 4 4 Nitrate B 0.1 25 Nitrite B 0.1

SS5 3.2 2 Nitrate A 0.08 25 Nitrite A 0.05

2.5.4 Preparation of Fortification Standard Spiking Solutions (Fort SS) Using a positive displacement pipet, transfer the volume of each source solution indicated in Table 4 into individual volumetric flasks. Dilute to volume with 10 mM sodium hydroxide solution. Mix thoroughly the solutions and store them at room temperature.

Table 4. Preparation of Fortification Standard Spiking Solutions

Solution ID Nitrate Target Concentration

(µg/mL)

Nitrite Target Concentration

(µg/mL)

Source Solution

Source Volume (mL) Final Volume (mL)

Fort SS1 100 100 Nitrate C 2.5

Nitrite C 2.5

Fort SS2 10 10 Fort SS1 5 50

2.6 Preparation of Calibration Standards (CS)

Using a positive displacement pipet, transfer the volume of each source solution indicated in Table 5 into individual volumetric flasks. Dilute to volume with 10 mM sodium hydroxide solution. Mix thoroughly the solutions and store them at room temperature.

Table 5. Preparation of Calibration Standards

Solution

ID

Nitrate Target Concentration in Solvent (µg/mL)

Nitrate Target Concentration in Tissue (mg/kg)

Nitrite Target Concentration in Solvent (µg/mL)

Nitrite Target Concentration in Tissue (mg/kg)

Source Solution

Source Volume

(mL)

Final Volume

(mL)

CS1 80 800 80 800 SS1 1 5

CS2 40 400 40 400 SS2 1 5

CS3 20 200 20 200 SS1 0.25 5

CS4 10 100 10 100 SS2 0.25 5

CS5 2 20 2 20 SS3 0.25 5

CS6 0.2 2 0.2 2 SS4 0.25 5

CS7 0.1 1 0.1 1 SS4 0.125 5

CS8 0.08 0.8 0.05 0.5 SS5 0.125 5

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2.7 Preparation of Homogenized Pig Liver

A butcher saw is used to cut frozen pig skeletal muscle into pieces no larger than approximately 50 grams. Each pig muscle sample is mixed approximately 1:1 (w:w) with dry ice and then homogenized using a food processor and/or food grinder until the tissue was homogenized to a powder. The dry ice was removed by sublimation at approximately -20°C. The homogenized pig muscle was transferred to a polypropylene container and stored frozen (-15 to -30°C) pending use.

2.8 Preparation of Fortified Pig Liver Samples

Using a positive displacement pipet, transfer the volume of each source solution indicated in Table 6 onto individual 3-g aliquots of bulk homogenized pig liver. Allow the samples to sit for approximately five minutes. Note: These samples are prepared fresh daily.

Table 6. Preparation of Fortified Pig Liver Samples

Sample ID Nitrate Target Concentration

(mg/kg)

Nitrite Target Concentration

(mg/kg) Source

Source Volume

(mL)

Tissue Weight (g)

Matrix 10x LOQ 33.3 33.3 Fort SS1 1 3 Matrix LOQ 3.33 3.33 Fort SS2 1 3

2.9 Preparation of Unfortified Pig Liver Samples

With each analysis set, process three unfortified 3-g aliquots of homogenized pig liver for use as unfortified matrix control samples.

2.10 Preparation of Centrifugal Concentrator Filtrate Receiver

Weigh the centrifugal concentrator filtrate receiver with the cap, but without the filter. Record the weight to a minimum of 4 significant figures on the centrifugal concentrator worksheet.

After the filtrate is collected in the centrifugal concentrator filtrate receiver, weigh the centrifugal concentrator filtrate receiver with the cap, but without the filter. Record the weight to a minimum of 4 significant figures.

2.11 Extraction and Processing of Unfortified and Fortified Pig Liver Samples

• If not previously weighed, weigh 3.000 ± 0.1 g of pig liver sample into a 50-mL polypropylene centrifuge tube.

• Using a graduated cylinder, transfer 30 mL of 10 mM sodium hydroxide solution to the 50-mL centrifuge tubes containing the pig liver samples.

• Place the samples on a rotator and rotate at a setting of approximately 60 for approximately 30 minutes. Rotate samples at approximately 2-8 °C.

• Centrifuge (J2-HS with J-17 rotor) the samples at a setting of ~ 7,500 revolutions per minutes (RPM) for 15 minutes, and at a temperature of approximately 2-8 °C.

• Transfer the supernatant to a clean individual 50-mL centrifuge tube.

• Weigh the centrifugal concentrator filtrate receiver with the cap, but without the filter.

• Transfer 15 mL of each supernatant to individual sample reservoirs of centrifugal concentrators.

• Centrifuge (GS-6R with GH-3.8 rotor) the samples at a setting of ~ 3,000 revolutions per minutes (RPM) for approximately 90 minutes, and at a temperature of approximately 2-8 °C.

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• Weigh the filtrate collected in the centrifugal concentrator filtrate receiver, without the filter.

• Transfer an aliquot of the filtrate to LC autosampler vials. Store the samples refrigerated (2 to 8 °C) pending LC analysis.

3.0 Analysis

Use the LC system conditions specified in Table 7. The conditions, which are designated, may be modified by the analyst to produce acceptable chromatography. Record the actual values inside the parentheses.

Set up the automated chromatography data software (CDS) system to collect the electronic output from each injection, acquiring the data.

Make a minimum of three injections of the SYS sample prior to starting the sample run to ensure optimal operation of the system.

Make single injections of all calibration standards, unfortified matrix control samples, fortified matrix samples, reagent blank, and carryover blank. Inject one calibration curve near the beginning of the run and one near the end of the run. The calibration standards near the beginning of the run will be injected from high to low concentration. The calibration standards near the end of the run will be injected from low to high concentration. One injection of the reagent blank will be injected prior to the first curve. A second injection of the reagent blank will be injected immediately following the second calibration curve.

The carryover blank will be injected, after the first, highest calibration standard. Distribute the unfortified matrix control blanks and the fortified matrix samples throughout the run.

Table 7. LC Analysis Conditions

Data System Thermo Scientific™ Dionex™ Chromeleon™ 7.2 LC Detection 215 nm

LC System Pump:_______________________________ Autosampler:_________________________ Detector: ___________________________

Analytical Column Dionex IonPac® AS11 (4 × 250 mm) (Thermo, Waltham, MA)

Guard Column Dionex IonPac® AG11 (4 × 50 mm) (Thermo, Waltham, MA)

Column Temperature (External) approximately 35°C Column Heater ID (External) Autosampler Temperature approximately 2-8°C

Mobile Phase A: 1 mM Sodium Hydroxide Solution B: 100 mM Sodium Hydroxide Solution

Mobile Phase Gradient

Time (minutes) 0.0

35.0 35.1 39.0 39.1 79.0

Eluent A (%)

Eluent B (%)

Flow Rate (mL/min) 1.0 mL/min Injection Volume 10 µL

Run Time approximately 79 minutes

Jon C. Andre (Battelle) Page 6 of 6 02October2020

4.0 Calculations

Determine the optimal automatic integration parameters for the run. Examine the integration of the analytes by the chromatography system. Modify, if necessary, to obtain optimum consistent integration.

Use Microsoft Excel to calculate the nominal concentration of each calibration standard.

Calculate the individual and average percent area of each blank compared with the average area response of the Lower Limit of Quantitation (LOQ) standard.

Calculate the individual percent area of the blank following the high standard compared with the area response of the nearest acceptable LOQ standard to assess carryover.

Calculate the regression equation relating the peak response ratio of each matrix standard (y axis) to the standard concentration (x-axis) using weighting if necessary. Evaluate both linear and quadratic regression models for at least one run. Start with a linear model with no weighting. Do not correct the response for experimental blank values. A data point may only be discarded after it has been shown to be statistically valid to do so.

Using the peak area response of the standards and the regression equation, and any dilution factor, if applicable, calculate the individual determined concentrations of the standards, blanks, and matrix samples.

Using the determined concentration, calculate the individual relative error (%RE) for each standard and fortified matrix sample concentration.

Calculate the individual and average determined concentration, the average RE, the standard deviation and RSD for the fortified matrix samples.