Experiment 3: Toxicants in Food

I. Introduction
o Toxicants in Food
o Natural origin as products of the metabolic processes of organisms (animals, plants, and microorganisms) from which the food is derived
o Biological and chemical contaminants from the environment (air, water, and soil)
o Intentionally introduced food additives
o Formed during the process of foods
o Salicylates
o Salt or ester of salicylic acid
o found in OTC medications and in numerous prescription drugs (e.g ASA)
Concentration (mg/dL)

Therapeutic range 15-30

Patients are often symptomatic >40-50

Serious or life-threatening toxicity Approaching or exceeding 100

Chronic poisoning with serious toxicity 60 or greater

o PK and PD:
▪ Absorption:
● Salicylates are readily absorbed in the unionized form from the small intestine (primarily from the jejunum and to a lesser extent, from the stomach and duodenum)
▪ Metabolism
● The major route of biotransformation is conjugation with glycine in the liver
▪ Excretion
● The amount of drug excreted unchanged in the urine is small but can be markedly increased if urine is alkaline
o Trinder Spot Test
o Rapid diagnostic test fpr salicylate exposure
o Trinder’s reagent (containing 10% ferric chloride) is made to react with a patient’s urine
o (+) = Violet color (complex salt of salicylic acid from its reaction with FeCl3
o Salicylates in food
o Found naturally in many herbs, vegetables, fruits, and nuts
o Act like preservatives to prevent rot and protect against pests
o Stored most vulnerable in eaves, barks, roots, skin and seeds

o Hypochlorites
o Salt or ester of hypochlorous acid
o Chloramines
o Derived of ammonia containing chlorine atom

o Hypochlorous and Chloramine in Milk

o Both can used as disinfectant in cleaning equipment and containers involved in processing of dairy products
o It is possible that certain amount of these toxicants are retained
o Chloramine Formation
NH3 + HOCl ⟷ NH2Cl (monochloramine) + H2O
NH2Cl + HOCl ⟷ NHCl2 (dichloramine) + H2O
NHCl2 + HOCl ⟷ NCl3 (nitrogen trichloride) + H2O
 o chloramine formation depends on the chlorine-to-ammonia ratio.

II. Methods
SALICYLATES IN BEVERAGES

Salicylic Acid Standard Solution

Alcoholic Sample

RFIS/ Important Notes:

1. NaOH - salicylate → sodium salicylate
● remove interferences present in alcoholic beverages which also affect FeCl3
● No addition of NaOH in non-alcoholic samples
2. 1:3 HCl - converts salicylate to salicylic acid
3. Salicylic acid will go to ether layer upon extraction.
Non-alcoholic Sample

RFIS/ Important Notes:

1. NaCl- demulsifying agent; increase ionic strength of liquid to break emulsion

Purification of Salicylic Acid

Method A Method B

HYPOCHLORITES AND CHLORAMINES IN MILK

Preparation of Solutions

Tests for Chloramine

III. Results and Discussion
Salicylates in Beverages
Ferric chloride Test
Qualitative test
Not specific for salicylates; other phenolic compounds may react with ferric chloride yielding false positive results
Reaction of ferric chloride with phenols is shown to exhibit a blue-violet colored solution due to the complex formed with Fe(III) and the hydroxyl group of the phenol
Salicylates are phenol derivatives and has an aromatic ring with an available hydroxyl group for formation of stable complexes with Fe(III).

Possible reasons of negative results

Undetectable level of salicylates in sample
Inefficient extraction of salicylates from the sample

Solution Ferric Chloride Test Indication

Standard (10% salicylic acid solution in water) Violet solution Presence of salicylates

Blank (Distilled water) Yellow solution or residue Absence of salicylates

Alcoholic beverage (Tanduay Ice Pomelo) Yellow solution Absence of salicylates

 Non-alcoholic beverage 1 (Water) Yellow solution Absence of salicylates

Non-alcoholic beverage 2 (Cobra energy drink) Red solution Absence of salicylates

Hypochlorites and Chloramines in Milk

Principle involved: Redox Reaction

Test I - Reaction with Potassium Iodide (7% KI solution) alone

2KI + Cl2 → I2 + 2KCl

Test II and III - Reaction with Potassium Iodide (7% KI solution) and 10% HCl solution
ClO- (hypochlorite) + 3I- + 2H+ ⟷ I3- + Cl- + H2O
NH2Cl (monochloramine) + 3I- + H2O + H+ ⟷ NH4OH + Cl- + I3-
NHCl2 (dichloramine) + 3I- + H2O + 2H+ ⟷ NH4OH + 2Cl- + I3-

● Triiodide ions in dilute solutions are yellow, more concentrated solutions are brown, and even more concentrated solutions are violet. Meanwhile, iodide solutions are colorless.

Test IV - Reaction with Starch Solution

2I3- (aq) + starch→ starch-I3-complex (blue-black color)

● Starch is used to indicate presence of triiodide ions even in small amounts.

Results
Solution Test I Test II Test III Test IV Concentration of Available
Chlorine

Standard (10% sodium Yellow color that disappears Light yellow solution Pale yellow solution Pale yellow solution ~ 1:5000
hypochlorite solution) upon shaking

Blank (Distilled water) Light yellow solution Light yellow solution Yellow solution Purple solution ~ 1:25000

Milk Unchanged milky white color White curd Flesh curd in pale yellow solution Flesh curd in pale yellow solution ~ 1:25000
with purple top layer
Note: Curd formation due to addition of acid resulting to the clumping together of casein in milk.

Interpretation of Results
Concentration of Available 1:1000 1:2000 1:5000 1:10000 1:25000
Chlorine

Test I Yellow brown Deep yellow Pale yellow

Test II Yellow brown Deep yellow Light yellow

Test III Yellow brown Deep yellow Yellow Dark yellow Pale yellow

Test IV Blue to purple Blue to purple Blue to purple Dark red to purple Red to purple