METHAMPHETAMINE

DETERMINATION IN URINE BY
REVERSED-PHASE HIGHPERFORMANCE LIQUID
CHROMATOGRAPHY WITH 1,2NAPTHOQUINONE 4-SULFONATE
AS DERIVATIZING AGENT AND
SOLID-PHASE EXTRACTION FOR
SAMPLE-UP
Nurul Afiera bt Md Ariffin (2014683038)
Nurul Fatehah bt Mohd Zailani (2014889052)

WHAT IS AMPHETAMINE?
Amphetamines are powerful, toxic and addictive,
central nervous system stimulants.

They are similar in chemical composition to

the body's natural stimulant adrenaline.

They are only legal when prescribed by a doctor

(often used to treat depression or obesity).

SHORT TERM EFFECT OF

AMPHETAMINE
Increased heart
rate/ blood

Increased
breathing

Loss of appetite

Increased in
energy

Memory loss

Aggression

WHAT IS METAPHETAMINES?
Methamphetamines are man-made
amphetamines.

They are usually made in home

laboratories from inexpensive ingredients.

Like most stimulant, it may induce strong

feelings of euphoria and can be addictive.

SHORT TERM EFFECT OF

METHAMPHETAMINE
Hallucinations

Alertness

Numbness

Confusion

Dangerous rise in
body temperature

Depression

INTRODUCTION
Identification and determination of Amphetamine
& methamphetamine in human urine sample by
Liquid Chromatography with UV-Vis detection.
Comment:

HPLC cannot detect many substance of interest

because they do not contain necessary
chromophoric, fluophroric or redox group.

Lack of sensitivity.

How to overcame this problem:

By using derivatization
Derivatization:
To permit analysis of compounds with inadequate
volatility or stability
To improve chromatographic behavior or detectability

OBJECTIVE
Amphetamine & methamphetamine determination in
urine by reversed-phase high-performance liquid
chromatography with 1,2-napthoquinone 4sulfonate as derivatizing agent. The usage of
reversed phase HPLC is due to its following ability:

Good sensitivity

Shorter time of separation.

SAMPLE TREATMENT

Extra-Sep C18 SPE columns were conditioned with 1.0 ml

methanol followed by 1.0 ml bicarbonate solution (pH= 10).
Urine sample (previously spike with amphetamine and
methamphetamine and 2.5 g/ml -phenylethylamine (I.S)
were drawn through the columns and subsequently washed
with 5 ml distilled water followed by 1 ml acetonitrile to
eliminate biological matrix.
The analytes and the I.S were eluted form the column with 2
ml methanol.

Why used SPE instead of LLE:

Save time (LLE usually 5x longer)
Simple
Provide high analytes recovery

WHY USED INTERNAL STANDARD?

I.S is known amount of compound, different from
the analyte, that is added to unknown sample.
The internal standard is a compound that is very
similar, but not identical to the chemical species
of interest in the samples.
Signal from analyte is compared with signal from
standard to quantify analyte.
It is used to correct for variability due to
analyte loss in sample storage and treatment.

WHY SPIKE THE SAMPLE WITH

AMINES STANDARD SOLUTION?
A known analyte with concentration gradient is
added to the sample.
->Signals of this known analyte is measured to
help us determine the concentration in the
original sample.

OPTIMIZATION

Different gradient elution condition were tested. (Hypersil ODS-C 18 column)

-To obtained the suitable sensitivity for detecting amines.
-Best result were obtained with mobile phase consisting acetonitrilewater (containing propylamine),
-To obtain resolution in shorter time.

The use of pH 10
- Good separation with sharp peaks.

RESULTS & DISCUSSION

Comment: pH 10 required only 10 minutes for the derivatization procedure

Comment: Peak analyte of interest become more sharp when spiked

with standard solution

Comment: High percent recovery for each analyte

The concentration were calculated from the calibration graph.

The precision is generally good.

VALIDATION
Limit of detection
The limit of detection was established by using the
standard solution procedure for signal-to-noise ratio
of 3 at a wavelength of 280 and 450 nm.

CONCLUSION
The chromatographic conditions were optimized to
obtain good sensitivity and separation of the analyte in
short time.

The use of SPE column save time in the sample

preparation and provide high analyte recoveries.

The use of reversed phase HPLC instead of normal phase

with UV Vis detection represent further improvement

RECOMMENDATION
The samples should be sent to other laboratories for
more accuracy and precision.
Increase the number of volunteers.
Taking your personal history (when take the test) to
avoid false positive result.