From the desk of Abhijit Bhar

Analysis of Phenols in Urine

The source of information about the state of our environment can be both the abiotic of the
environment (water, Soil, Air) as well as samples of the biotic part, including tissues and body fluids of
humans, who are continuously exposed to a wide spectrum of xenobiotic chemicals. We can distinguish
three fundamental kinds of exposure through which environmental pollutants can enter in human body.

Endemic exposure – Associated with human contact with drinking water, indoor air, outdoor air and
food ingestion.

Workplace exposure – Associated with performing specific operations and activities at the workplace.

Catastrophic exposure – Associated with exposure to pollutants resulting from unexpected event
(ecological disasters).

Xenobiotic chemicals can enter the human body via three paths: through skin, through respiratory
system and through the digestive system. Depending on their physicochemical properties, these
chemicals take different paths in the body. A fraction of them is cumulated in the tissues (bone, adipose
and parenchymatous) and the organs (brain, liver, kidneys), while the rest undergoes form with the
expelled air, bile, urine, saliva or sperm. Renal excretion plays the greatest role in higher organisms.

The analysis of samples taken from humans presents a great analytical challenge. In order to determine
metals and , most of all, a variety of organic compounds in such samples, they must be subjected to
tedious and time consuming sample preparation procedures. Samples of physiological fluids (including
urine)have very complex matrices and in the majority of cases, direct determination of analytes by
means of commonly used methods and techniques is impossible.

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From the desk of Abhijit Bhar

Irrespective of the way a chemical compound enters the body (except for intravenous injection), after
penetrating the initial cellular barrier the compound first reaches the intercellular fluid. From there the
compound penetrates capillary blood vessels and thus enters the circulatory system, which distributes it
throughout the entire body. A majority of toxic compounds do not cause damage at the site where they
enter the body.

Biotransformation and elimination of poisons takes place primarily in the liver and the kidneys. Toxins
absorbed in the digestive tract are transported in blood from the stomach and the intestines to the liver.
The liver is the site where the absorbed toxins undergo rapid transformation involving enzymes, which
are especially abundant there. Some substances become soluble in water and are transported with
blood to the kidneys. Once there, they are filtered, removed from blood as polar toxins and metabolites,
and then excreted with urine. For example, Benzene is metabolized in the liver to phenolic compounds,
which are excreted in urine along with other metabolites. The determination of specific metabolites in
urine is often used as a quantitative indicator of exposure to a specific substance. This process is called
“biomonitoring”, and the metabolite itself is called a “biomarker” or “bioindicator”. Benzene undergoes
metabolic transformation to phenol, which poisons bone marrow.

To analyze the phenol in urine (as biomarker), it needs to extract it from the matrices. As, urine consist
of 95% water along with products of metabolism (mainly urea), protein, glucose, ketones, etc; it is
important to take care of a method of extraction of phenol with a good reproducible in terms of
qualitative and quantitative parameters. Phenol in Urine can be analyzed by GC-FID or GC-MS after
extraction by liquid-liquid extraction, Microwave extraction or by using Headspace. Among of these,
Headspace is most suitable technique as it requires minimum sample preparation, minimize secondary
contamination through solvents and possible of direct analysis of samples without diluting by solvents.

Liquid phase extraction (LLE) is still widely using despite of several disadvantages as it has a high
detection limit due to large sample volumes uses for extraction. Main disadvantages is the need of large
amount of organic solvents which to be evaporated to concentrate for target molecules.

Sample preparation:

Method – 1

1. Pipet 5ml urine in to a 15ml centrifuge tube

2. Add 1ml conc. HCl or 5 drops 70% perchloric acid. Mix. Well.
3. Stopper loosely. Heat in a water bath at 950C for 1.5 hrs.
4. Remove from water bath. Add 10 ul internal standard (dissolve 30mg nitrobenzene in 50ml
methanol). Adjust volume in the centrifuge tube to 10 ml with distilled water.
5. Pipette 2ml diethyl ether and 1gm NaCl in to the tube. Stopper and shake vigorously for 1 min.
Cool the tube to 00C and allow the phases to separate.

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From the desk of Abhijit Bhar

Method – 2

1. Take 1ml of urine.

2. Mixed with 0.5ml 15% HCl, heat for 1 hr for acid hydrolysis.
3. Add 5ug 3,5-xylenol as internal standard.
4. Extract the mixture with 2ml Carbon disulfide.
5. Treat it with sodium sulphate for desiccation.
6. Take 1ml aliquot to a test tube and reduce the volume to about one twentieth or less under a
stream of nitrogen.
7. Inject 1 ul to GC.

For the hydrolysis, different methods are telling to use different acids and to extract different solvents.
Bellow is some references:

1. Phenolic acid (Balikova and Kochlicek, 1989)

2. HCl (NIOSH, 1984; Ong et al 1988)
3. Sulfuric acid (Pierce and Nerland, 1988; Rick et al 1982)
4. Perchloric acid (Baselt 1980; NIOSH 1984; Rick et al 1982)
5. Enzymatic methods (Buchet 1988).

Enzymatic method differ from others in respect to the time necessary for the hydrolysis, since 18hours is
used with enzymes and 30 – 60 min with acid hydrolysis.

Extraction can be done with following solvents:

1. N-hexane (Balikova and Kohlicek, 1989)

2. Diethyl ether (Buchet 1988; NIOSH 1984, Ong et al 1988)
3. Ethyl acetate (Pierce and Nerland 1988)
4. Diisopropyl ether (Baselt 1980; NIOSH 1980, Rick et al 1982)
5. Methyl tert-butyl ether (Rick et al 1982).

Chromatographic condition:

- Instrument :GC with FID

- Column: 100% methyl, 15mt x 0.53mm ID x 1.5 micron FT
- Injector temp.: 2000C
- Detector (FID) temp.: 2000C
- Oven temp.: 600C, hold for 2min, ramp to 1200C by 100C/min

Note: Selection of column length, ID and film thickness may need to change if other components of
interest are there.

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