FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

SUBJECT FORENSIC SCIENCE

Paper No. and Title PAPER No.12: Forensic Biology and Serology

Module No. and Title MODULE No.10: Biological Characteristics, Identification,

General Test and Analysis of Drug in Urine

Module Tag FSC_P12_M10

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

TABLE OF CONTENTS

TABLE OF CONTENTS

1. Learning Outcomes

2. Introduction: Urine

3. Constituents of urine

4. Purposes of urine test

5. General Urine test

6. Test of degraded protein in urine

6.1 Bilirubin

6.2 Urobilinogen

6.3 Bench jones proteins

7. Confirmatory test of urine

7.1 Detection of urea by Gee’s urea nitrate test

7.2 Detection of Creatinin

7.3 Detection of Indican

8. Analysis of drug in Urine

9. Presumptive substance testing

10. Diagnostic screening of urine

11. Determination of time for different drug in Urine

12. Forensic detection of drug Marijuana in Urine

13. Summary

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

1. Learning Outcomes

After studying this module, you shall be able to know-

What is Urine?

What are the constituents of Urine?

What are the purposes of urine testing?

What are the confirmatory tests of Urine?

What is a diagnostic screening of Urine?

How we will do the alcohol test through Urine?

2. Introduction: Urine

Urine can be defined as the liquid waste product secreted by kidneys by a process known

as urination and is excreted through the urethra. Cellular metabolism produces various

waste products that are needed to be cleared from the bloodstream and are also rich in

nitrogen. Healthy urine is not toxic and only undesirable substances are eliminated by the

body. Usually the urine is transparent pale yellow solution but its color also ranges from

colorless to pale yellow. Over-hydration is indicated by the presence of colorless urine.

During the process of drug testing if colorless urine is detected then it suggests an attempt

to avoid the detection of illegal drugs in the bloodstream through over-hydration.

Examination of urine may sometimes require in the investigation of unnatural death like

hanging and strangulation. A microscopic examination of the urine may be helpful to

identify organic or inorganic substrates that help in the diagnosis. Many tests are used in

analyzing the urine in order to determine, whether it contains abnormal substances

indicative of diseases. Usually the substances that are not present in urine and can only be

detected during its analysis are glucose, blood, protein, acetone and pus. Some renal

function tests are based on clearance of metabolites into the urine.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

3. Constituents of Urine

NORMAL CONSTITUENTS

ABNORMAL CONSTITUENTS

Sodium chloride, uric acid, hippuric acid,

potassium, oxalate, amino acid, ammonia,

traces of glucose, ketose, vitamins, hormones,

calcium, sulphate and keratinine.

Sugar, protein, ketone bodies, bile salts,

etc.

Contents and appearance

The color of normal urine is that of clear straw and consists of slight odor with some

crystals and small number of cells from the tissues which line the urinary bladder. Sugars,

proteins, bacteria, yeast cells, ketones and parasitic organisms are not present in normal

urine.

A different time in a day a urine sample is collected can make a difference in the

appearance of the specimen. Medicines and foods such as penicillin and red beets,

asparagus may affect the odor and color of urine. Although most color variations are

harmless, they sometimes indicate the presence of serious disease.

4. Purpose of Urine Test

A urine test may be done:

To check for a disease or infection of the urinary tract. The various signs and

symptoms shown in case of an urinary infection consists of colored and bad-

smelling urine, pain and finding it hard to urinate, blood in urine, etc.

In treatments for kidney stones, diabetes, UTI i.e. urinary tract infection, high BP,

urine tests are done so as to check for the conditions.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

To diagnose the diseases or disorders of the kidneys or urinary tract and

monitoring of patients having diabetes.

To test for pregnancy

To screen for drug abuse

In a urine test, waste by-products made by the kidneys and the various components

present in it are checked for.

5. General Urine Test

Colour Normal: Pale to dark yellow in colour

Abnormal: Various foods and medicines and drugs may affect the urine

color. Long-term kidney disease or uncontrolled diabetes may also result in

color change of urine. Dark yellow urine can be caused by the dehydration.

Blood in urine results in its red color.

Clarity Normal: Clear

Abnormal: Cloudy urine can be caused by pus, blood, sperm, bacteria,

yeast, crystals, mucus, or a parasite infection, such as trichominiasis.

Odour Normal: bad odour

Abnormal: Consumption of some food, vitamins, and antibiotics can cause

urine to have a different odour. A sweet, fruity odour may be caused by

uncontrolled diabetes

Specific gravity

Normal: 1.005–1.030

Abnormal: A very high specific gravity means very concentrated urine,

which may be caused by, loss of too much fluid, sugar or protein in the urine.

Very low specific gravity means diluted urine, which may be caused by

drinking too much fluid or severe kidney diseases.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

pH Normal: Ph between 4.6–8.0

Abnormal: Some foods and medicines can affect urine pH; it can be caused

by severe vomiting, a kidney disease, some urinary tract infections, and

asthma. A low pH may be caused by severe lung diseases.

Protein Normal: None

Abnormal: Protein in the urine indicates kidney damage, an infection,

cancer, high blood pressure, diabetes.Protein in the urine may also indicate

that the person is suffering from leukemia.

Ketones Normal: None

Abnormal: Ketones in the urine shows uncontrolled diabetes, a very low-

carbohydrate diet, starvation or eating disorders.

Microscopic

analysis

Normal: Very few or no red or white blood cells or casts are seen. No

bacteria, yeast cells, parasites, or squamous cells are present. A few crystals

are normally seen.

Abnormal: Red blood cells in the urine indicate kidney or bladder injury,

kidney stones, a urinary tract infection (UTI), rupture of blood vessels in

kidney.

6. Test of Degraded Protein in Urine

Urine samples are tested with different instruments and techniques. In some cases

dipsticks are used, i.e. strips of plastic which changes color in presence of particular

substances. Dipsticks can be used to measure the acidity of the urine (its pH) or the

presence of blood, protein, sugar, or substances produced during the breakdown of fatty

acids. Examination of the urine specimen is also carried out with the help of a microscope

so as to determine if it contains crystals, pieces of fibrous material and even blood cells.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

The various components that are checked for during a urine testing includes:

6.1 Bilirubin

Bilirubin is a bile salt formed by the breakdown of red blood cells and is passed on from

the body in feces. Generally, urine does not consist of bilirubin. If it is present, it means

that the liver is damaged or that the flow of bile from the gallbladder is blocked and

passes in blood streams. During the process of normal heme catabolism, the final

breakdown product results in the yellow colored bilirubin. Heme is present in

hemoglobin, the main component of the red blood cells.

Bile and urine shows presence of bilirubin but its elevated level may indicate the

presence of various diseases. It is responsible for the yellow color of bruises, the

background straw-yellow color of urine via its reduced breakdown product, urobilin the

brown color of feces, and the yellow discoloration in jaundice. It is also present in plants.

Bilirubin has an open chain structure of four pyrrole-like rings and in case of heme

molecule; the four rings are connected to a larger ring known as the porphyrin ring. It can

be made soluble in water by conjugating it with glucuronic acid and this process is an

example of glucuronidation. Also, the substance bilirubin is very much similar to the

phytochrome pigment found in plants that are used to sense light and also to the pigment

phycobilin used by particular algae to capture light. All these components have an open

chain structure consisting of four pyrrole rings. On being exposed to light, some of the

double bonds present in bilirubin tend to isomerize. Thus, this property is exploited in the

technique of phototherapy used for the jaundiced newborns wherein the E, Z isomers of

bilirubin formed due to the exposure of light is more soluble than the un-illuminated Z

isomer because there is removal of the intermolecular hydrogen bonding thereby,

allowing the removal of the unconjugated bilirubin in the bile.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

Bilirubin is formed by the action of the biliverdin reductase on biliverdin which is a

product formed during the process of heme catabolism resulting in a green colored bile

pigment having a tetra-pyrrolic structure. On oxidation of bilirubin, it again gets

converted into biliverdin. Because of this process and the demonstration of the effective

antioxidant property of bilirubin, it has resulted in the hypothesis that bilirubin's main

physiologic role is as a cellular antioxidant. Bilirubin is degraded by light and thus, to

carry out bilirubin assays serum and blood samples should be protected from

illumination. For adults, blood is typically collected by needle from a vein in the arm.

Blood is collected with the help of a heel stick in infants wherein a sharp, small blade is

used to cut the heel and thereby few drops of blood are collected in a small tube. The

Bilirubin level found in the body reflects the balance between production and excretion.

Blood test results should always be interpreted using the reference range provided by the

laboratory that performed the test, but typical [0.3 to 1.9 mg/dL] s for adults and

[340µmol/L] for new born.

The circulation of bilirubin in the bloodstream is carried out in two forms:

Indirect Bilirubin. The indirect form of bilirubin is not soluble in water but once

it travels through the bloodstream into the liver, it is changed into the soluble

form.

Direct Bilirubin. It is formed from the indirect bilirubin in liver and readily

dissolves in water.

The measurement for direct bilirubin levels and the total bilirubin can be measured from

the blood but the indirect level of bilirubin is derived from the direct and total bilirubin

measurements. High levels of bilirubin cause the whites of eyes and the skin to appear

yellow. When bile passes from the liver to the small intestine, it causes blockage of tubes

and various blood disorders and jaundice may be caused. In infants, minor jaundice may

not pose serious problems but high bilirubin levels results in brain damage and other

serious issues.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

Bilirubin tests are carried out to:

Check liver damage caused due to various medicines and also in cases of liver

disease for example, cirrhosis and hepatitis.

Find out the presence of tumors in pancreas or the presence of gallstones that may

block the bile ducts.

Diagnose conditions that cause increased destruction of red blood cells, such as

hemolytic anemia or hemolytic disease of the newborn.

Help make decisions about whether newborn babies with neonatal jaundice need

treatment. These babies may need treatment with special lights, called

phototherapy. In rare cases, blood transfusions may be needed.

6.2 Urobilinogen

Urobilinogen is formed by the breakdown of bilirubin. It is also passed from the body in

stool but only small amount of it is present in urine. High amount of Urobilinogen may be

a sign of disease caused in liver or it may also be that the flow of bile has been blocked

from the gallbladder.

6.3 Bence Jones protein

In about 50% individuals suffering with a rare type of cancer, this abnormal protein is

found in their urine. A urine test is often done when multiple myeloma is suspected.

7. Confirmatory Test for Urine

The confirmatory stain test are performed for the detection of various chemical

constituents present in urine like urea, creatinine and indican.The common stain are

7.1Detection of Urea by Gee’s Method

The suspected stain is extracted with acetone and then the acetone extract is concentrated

by evaporation. The concentrated extract is then filtered and then it is evaporated to

dryness.

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PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

The residue is dissolved in acetone mixed with the help of glass rod. A drop of the

solution is then taken on a microscopic slide and then again it allowed to evaporate, after

this the separation of the urea crystals may be observed. The crystals are long colourless,

4 or 6 sided rhombic crystals. The nitric acid solution is added by the help of glass rod,

the urea nitrate crystals may be observed.

7.2 Detection of Creatinine

Concentrated stain is taken on a chromatographic paper and it is treated with 2N NaOH

solution and then 1 drop of picric acid. The red color crystal indicates the presence of

creatinine.

7.3 Detection of Indican

1 ml of resorcinol reagent is added to the small quantity of the extracted stain then the

1ml of cupric bromide solution is added mixed and whole mixture is extracted with

amylacetate.The red colour of the crystal indicates the presence of Indican.

8. Drug Screening in Urine

Screening of drug test in the urine is done for the presence of sedative parent drug or its

metabolites. The drug level and their metabolites are not predictive as when the drug was

taken or how much it was taken. It is simply a confirmatory test indicating the presence

of the parent drug or its metabolites. The collection for urine sample is done at a remote

location in a specially designed cup sealed with tamper-resistant tape and dispatched to a

testing laboratory to be screened for drugs.

9. Presumptive substance Testing

The presumptive tests are carried out so as to identify a suspected material or substance.

This test involves the mixing of the suspicious material with a chemical in order to a

color change that indicates if a drug is present. A very small amount of material is needed

to obtain results, and can be used to test powder, pills, capsules, crystals, or organic

material.

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PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

This is also indicative to detect illicit material when mixed with other non-illicit

materials. The tests are used for general screening purposes, offering a generic result for

the presence of a wide range of drugs, including Heroin, Cocaine (Crack),

Methamphetamines, Amphetamines, Ecstasy/MDMA, Methadone, Ketamine, PCP,

PMA, DMT, MDPV, Mephadone, and may detect rapidly evolving synthetic designer

drugs in urine.

10. Diagnostic Screening in Urine

This test is used in the cases of life-threatening symptoms, unconsciousness or in an

emergency situation, screening for common drugs .These tests can also be done post-

mortem during an autopsy in cases, where a death was not expected. The test is usually

done within 96 hrs after the desire for the test is realized. Both a urine sample and a blood

sample are tested. Various panels are used for screening urine samples for common

substances, e.g. triage 8 that detects amphetamines, benzodiazepines, cocaine,

methadone, opiates, cannabis, barbiturates and tricyclic antidepressants. Results are

observed in 10–15 min.Similar screenings may be used to evaluate the possible use of

date rape drugs. This is done in urine sample.

11. Determination of Time for Different Drugs in Urine

Types of drugs Detection time in urine

Marijuana - Single Use 1-7+ days

Marijuana - Regular Use 7-100 days

Amphetamines 1-3 days

Cocaine 1-3 days

Heroin, Opiates 1-4 days

PCP 3-7 days

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

The most popular drug test is the urine test, by which the drug marijuana can be detected

for days or weeks even after its use. Though these tests are not very effective in detecting

the psychoactive compound THC i.e. delta-9-tetrahydrocannabinol present in the drug

marijuana, but they detect the non-psychoactive marijuana metabolite THC-COOH, that

lingers in body for days or even weeks with no prejudicing effects. As the non-

psychoactive component has an unusual longer elimination time, that’s why these tests

are more sensitive to marijuana as compared to other commonly used drugs.

12. Forensic Detection of Drug Marijuana in Urine

The alcohol test is carried out so as to determine whether the individual has been drinking

in the past or not. However these tests are not appropriate to determine whether or not

someone is currently intoxicated, because a positive outcome does not necessarily

indicate the immediate presence of alcohol in the body. Urine alcohol Tests, including

EtG tests, are typically used in situations, when the person is required to be completely

abstinent, either permanently, or over a specified period of time. This can be contrasted to

situations where people are permitted to drink legally while on their own time, but not

permitted to drink while performing specific task. In this case, an alcohol urine test at the

conclusion of the period in which he or she has visitation or custody could be

administered to ensure that he or she did not consume alcohol in the recent past. Urine

alcohol tests are frequently required by court order or signed agreement between former

spouses.

FORENSIC SCIENCE

PAPER No.12: Forensic Biology and Serology

MODULE No.10: Biological Characteristics, Identification, General Test and Analysis of Drug in Urine

13. Summary

Usually the urine is transparent pale yellow solution but its color also ranges from

colorless to pale yellow.

The color of normal urine is that of clear straw and consists of slight odor with

some crystals and small number of cells from the tissues which line the urinary

bladder.

Examination of the urine specimen is also carried out with the help of a

microscope so as to determine if it contains crystals, pieces of fibrous material

and even blood cells.

Bilirubin is formed by the action of the biliverdin reductase on biliverdin which is

a product formed during the process of heme catabolism resulting in a green

colored bile pigment having a tetra-pyrrolic structure.

Urobilinogen is formed by the breakdown of bilirubin.