Introduction

Arsenic in Biological Materials

Once referred to as ‘the inheritor’s powder’, arsenic was a popular poison choice (in addition to being used in make-up and complexion products). Now, however, arsenic exposure routes in the U.S. are typically accidental through ingested food or inhalation exposure in some workplace environments. In other parts of the world, contaminated drinking water is also a major route of exposure.

As discussed in class, there are inorganic and organic arsenic-containing compounds that may be of interest and that vary in toxicity. Most often, total arsenic (total concentration of all arsenic species) is analyzed in urine. However, liver samples can sometimes be useful in complex poisoning cases, so we’ll learn how to digest tissue organ tissue for toxicological analysis in this lab.

Sample Preparation

To analyze a liver sample, we first must perform a tissue digestion, or essentially dissolve the tissue. Depending on the type of tissue and the end goal, there are many ways one might accomplish this. We will use a concentration HCl solution and then filter our sample prior to analysis.

We will also need external calibration solutions of known arsenic concentration so we can calculate the total arsenic concentration in our sample. To prepare these, you’ll have a standard solution that contains 10 ppm arsenic in 2% nitric acid.

It is important that you use ultrapure water, or water with a resistivity of 18.2 M

$\mathrm{\Omega }$

, for any dilutions or sample preparation for mass spectrometry analyses. Ultrapure water undergoes multiple types of purification to remove contaminants of the ionic, organic, bacterial, and solid varieties. It is important to understand that this is not the same as deionized water.

Analytical Techniques

Atomic absorption spectroscopy (AAS), inductively-coupled plasma – atomic emission spectroscopy (ICP-AES), and inductively-coupled plasma – mass spectrometry (ICP-MS) are all potential analytical methods for the determination of arsenic in biological materials. If differentiation of arsenic species is necessary, an in-line separation method would be used (like liquid chromatography or capillary electrophoresis) to separate them prior to analysis.

In this exercise, will use ICP-MS to determine total arsenic. ICP uses a plasma to ionize and atomize the sample. It is a common technique for analyzing many metal species and some non-metals. You’ll also get a brief look at a quadrupole mass analyzer. The mass analyzer is the component of the mass spectrometer that separates ions of different mass-to-charge charge ratios so that they arrive at the detector at different times. There are several types of mass analyzers with the quadrupole being one of the most popular.

Materials and Methods

Supplies (Part 1)

1. Liver sample
2. Flask or other vessel to hold digesting tissue
3. Concentrated nitric acid
4. Vessels and pipettes for calibration solutions

Procedure (Part 1)

Begin Tissue Digestion

1. Place 500 mg of tissue in vessel with 5 mL of concentrated nitric acid
2. Place vessel in digester
3. This will digest until the next lab day

Prepare Calibration Solutions

1. Using the glassware and pipettes provided, calculate how to make the following arsenic solutions from 10 ppm stock.
   • 0 ppb
   • 1 ppb
   • 5 ppb
   • 10 ppb
   • 20 ppb
   • 30 ppb
2. Check with a TA to ensure you’ve performed this correctly.
3. Store solutions for the next lab day.

Supplies (Part 2)

1. Tissue digestion flask
2. Tissue digester
3. 18 M
   $\mathrm{\Omega }$ 

   water

4. Syringe filter
5. Calibration solutions
6. ICP-MS

Procedure (Part 2)

Finish Tissue Digestion

1. Heat tissue sample for 20 minutes at 165
   ${}^{\circ }$ 

   C

2. Cool to room temperature
3. Add 45 mL of 18 M
   $\mathrm{\Omega }$ 

   water

4. Filter with syringe filter

Analysis

1. Run calibration solutions in order with TA assistance
2. Once calibration data is deemed acceptable, run sample solution
3. Obtain one spectrum CSV
4. Use calibration data to determine total arsenic in the original sample