International Journal of Pharmaceutical Sciences

International Journal of Pharmaceutical Sciences INT.J.PH.SCI.,SEP-DEC, 2010;2(3):771-776 ISSN 0975-4725 www.ijps.info

Original Research Manuscript Date of Submission: 30-08-2010 Date of Acceptance: 06-11-2010

IMPURITIES RESPONSIBLE FOR CHANGE IN PHYSICAL CHARACTERISTICS OF COMPRESSED PARACETAMOL TABLETS NELSON A. OCHEKPE AND AYODEJI A. AGBOWURO. Department of Pharmaceutical Chemistry, University of Jos, Jos, Nigeria

ABSTRACT For some years now, there have been some occasional reports of compressed Paracetamol tablets getting dark on the shelf and shortly after compression. The purpose of this investigation is to identify the chemical impurities responsible for this observation in some batches of Paracetamol produced in a private manufacturing out-fit. The impurities would be isolated and characterized using readily available and relatively simple methods. Possible degradation route(s) would be proposed as well as ways to minimize the observed degradation. Methanolic extracts of darkened paracetamol tablets and that of a reference standard paracetamol tablet were subjected to a gas chromatography – mass spectrometric (GC-MS) analysis. Isolation of some of the impurity products was carried out using accelerated gradient chromatography (AGC), GC-MS, while the fractions were exposed to infrared spectroscopy (IR). At every stage confirmatory thin layer chromatographic procedures were used to monitor and confirm the identities of products isolated and characterized. The gas chromatographic spectra of the test samples when compared to that of the reference standard revealed several other peaks, two of which were identified by the coupled mass spectrometer to be N-phenyl glucosamine and 4-aminophenol which were also confirmed by Thin Layer Chromatography (TLC). A fraction obtained from AGC was characterized by IR and GC-MS to be a mixture of paracetamol and aniline. The results suggest a base-catalyzed hydrolysis of paracetamol to 4-aminophenol which was further degraded to aniline, the formation of N-phenyl glucosamine, which is a black compound confirmed a Maillard-Amadori reaction between aniline and a reducing sugar probably the lactose used as a bulking agent. Keywords: Darkened Paracetamol, GC-MS, hydrolytic, Maillard-Amadori reaction.

volatile or non-volatile and may include: starting

INTRODUCTION Impurities

in

pharmaceuticals

are

the

unwanted

materials, by-products, degradation products.

chemicals that remain with the active pharmaceutical

Degradation

ingredients (APIs), or develop during formulation, or

formulation to different dosage forms or ageing are

upon ageing of both API and formulated APIs in

common impurities in medicines. The degradation of

medicine(1). According to the International Conference

penicillin and cephalosporins is

on Harmonization (ICH)(2) organic impurities may arise

classical textbooks on the topic. The presence of a â-

during the manufacturing process and/or on storage of

lactam ring as well as that of an á-amino group in the

products

resulting

from

storage

or

well documented in

the drug substance, it may be identified or unidentified,

771

Int.J.Ph.Sci.,May-August 2010;2(3):

NELSON A ET AL: IMPURITIES RESPONSIBLE FOR CHANGE IN PHYSICAL CHARACTERISTICS C6/C7 side chain plays

a critical role in this

MATERIALS AND METHODS

degradation(3).

Chemicals and Reagents:

It is often necessary to isolate degradation products.

Analytical

Generally, chromatographic and non-chromatographic

concentrated

techniques are used prior to their characterization.

paracetamol reference standard were obtained with the

Among the chromatographic methods used are TLC,

MiniLab® from Global Pharma Health Fund (GPHF), 4-

GC, AGC and high performance liquid chromatography

aminophenol was purchased from Merck. Darkened

(HPLC) (4).

paracetamol Samples were supplied by a pharmaceutical

Paracetamol (acetaminophen) is an over the counter

grades

of

methanol,

ammonium

Ethyl

hydroxide,

acetate, authentic

manufacturing company in Nigeria.

5

antipyretic analgesic agent that is widely used . It is synthesized by nitrating phenol with sodium nitrate,

Equipment:

separating the desired para-nitrophenol from the ortho-

Equipments and apparatus used included; GCMS-

byproduct and reducing the nitro group with sodium

QP2010 PLUS Shimadzu, Shimadzu FTIR spectrometer,

borohydride. The resultant para-aminophenol is then

Bækstrom AGC equipment, Rotary evaporator, Pestle,

acetylated

Industrial

Aluminum foil, laboratory glass bottles with a filling

conversion of paracetamol powder to tablets involves

capacity of 25 to 100ml, funnel, set of straight pipettes

dry or wet granulation, the latter involves mixing

(1 to 25ml), 10ml vials, Merck TLC aluminum plates

paracetamol drug powder with some quantity of water,

pre-coated with silica gel 60F254, size 5 X 10 cm, glass

drying it at a specified temperature and compressing the

micro-capillaries of 2µl filing capacity hot plate, TLC

resulting granules with other materials called excipients.

developing chamber, filter paper, and a pair of scissors.

with

acetic

anhydride(6).

Some of the common impurities present in paracetamol drug powder include 4-aminophenol, chloroacetanilide

EXTRACTION,

and

CHARACTERIZATION

4-nitrophenol

and

according to the

British

AND

Pharmacopoeia, none of the limits of these impurities

30 tablets of darkened paracetamol (Fig 1) of an average

should exceed 0.1 percent(7).

weight of 650mg were pulverized. The powder was

There have been several documented and

772

ISOLATION

stepwise extracted with aliquots of methanol. The extract

undocumented reports of paracetamol tablets getting

obtained was concentrated on a rotary evaporator.

darkened on the shelves in pharmaceutical outlets and

Accelerated gradient chromatography of the methanol

shortly after production. As a result of this problem

extract was carried out using 50ml n-hexane and

several pharmaceutical companies have lost so much in

gradually increasing the proportion of ethyl acetate till a

form of money and reputation. The samples used in this

mixture of ethyl acetate-methanol of 50:50 on silica gel

study were supplied by a pharmaceutical manufacturing

G was achieved. Based on their resolutions in ethyl

company that is a leading producer of paracetamol

acetate-methanol 8:1 (V/V) on TLC silica gel GF254, a

tablets in Nigeria. It is part of good cGMP to have

unique fraction labeled ISOL-2 was subjected to IR

research and thorough investigations carried out on any

spectroscopy. The IR spectra were registered on a

defects found pre- and post-marketing of products so as

Shimadzu FTIR spectrometer using KBr discs (samples

to prevent reoccurrence and to improve manufacturing

were treated as solids prepared using the “cast film”

practices. Investigations such as this also help to further

method)(8).

the frontiers of Pharmacovigilance and may help to

isolates and the crude extracts using the guidelines

explain issues of bioequivalence of medicines.

modified by A Danell(9).

GC-MS analysis was carried out on the


NELSON A ET AL: IMPURITIES RESPONSIBLE FOR CHANGE IN PHYSICAL CHARACTERISTICS carbon double bond (1515 cm-1), Ar-H stretching was

THIN LAYER CHROMATOGRAPHY of

indicated by absorption at 2984.94 cm-1. The presence in

paracetamol by thin layer chromatography developed by

the fingerprint region of (633 – 733 cm-1) confirmed a

A. S. Kenyon and T. P. Layloff(10) was employed. A

mono substituted ring. EIMS (70ev, 200oC) M/Z: 93

darkened paracetamol tablet labeled sample C and a

[M]+, 77 [M_NH2]+, 65, 51, 40.

parecetamol reference standard tablet labeled sample E

B)Paracetamol, retention time of 27.6 min; the IR

were crushed in aluminum foil using a pestle; each was

spectrum indicated the presence of an aromatic broad

extracted with 10ml methanol and filtered. 2µl of each

OH band (3453.66 cm-1), carbonyl group (1896 cm-1)

were spotted on the chromatoplate alongside a solution

while 1097.53 cm-1 band indicated the C-O bending.

of 4-aminophenol in methanol labeled AP using micro-

Aromatic carbon-carbon double bond (1515 cm-1), Ar-H

capillaries, the plates were developed in a chamber

stretching was indicated by absorption at 2984.94 cm-1,

containing 24ml ethyl acetate, 3ml methanol and 1ml

1242.2 cm-1 is characteristic of OH bending. Sharp bands

concentrated ammonium hydroxide. The spots were

obtained in the fingerprint region (846-794 cm-1)

detected by viewing the plates under UV light of 254 nm

confirmed the presence of 1,4-aromatic di-substitution.

wavelength.

EIMS (70ev, 200oC) M/Z: 151 [M]+, 122, [M_COCH3]+

The

unofficial

method

for

rapid

screening

109, 91, 80, 65, 42. RESULTS The gas chromatogram of a methanolic extract of the

DISUCSSION

darkened paracetamol tablets (Fig 2) when compared to

The impurities isolated from the darkened paracetamol

that of the reference standard revealed several other

tablets suggest a base-catalyzed hydrolysis of the bulk

peaks two of which were characterized by the coupled

paracetamol powder and prior contamination of starting

mass spectrometer based on their fragmentation patterns

raw materials. Paracetamol in a basic environment was

to be: Compound 1 (Retention time: 30.8 min.), 4-

hydrolyzed by water from residual moisture of wet

o

+

aminophenol with EIMS (70eV, 200 C) M/Z: 109[M] ,

granulation and/or moisture sorbed into excipients such

80, 64, 53, 39; and Compound 2 (Retention time: 31.5

as starch and lactose to 4-aminophenol which is an

o

min.), N-phenylglucosamine with EIMS (70eV, 200 C) +

M/Z: 253[M] , 208, 185, 129, 112, 98, 69, 55.

The formation of glucosamines which are known black

Thin layer chromatography of the methanolic extract

compounds responsible for the observed physical color

alongside 4-aminophenol and paracetamol reference

change in the tablets confirmed a Mallard-Amadori

standard confirmed the presence of compound 1 with a

reaction; this is a reaction between reducing sugars and

retention factor (RF) of 0.91 (Fig 3).

primary amines and is responsible for changes in color,

An attempt to isolate each compound by accelerated

flavor and nutritive in food. This reaction is initiated by

gradient chromatography was limited by the similar

the formation of N-glycoside followed by an Amadori

solvents’ solubility displayed by the degradation

rearrangement to a compound called ketosamine ended

products and paracetamol. However, a unique isolate

by a dehydration step(11) with loss of water molecule

labelled ISOL-2 on infrared spectroscopy and GC-MS

(Fig 4).

was discovered to be a mixture of two compounds:

To minimize this degradation, it is important to as much

A) Phenylamine (aniline), retention time (3.9 min); the

as possible minimize the moisture content of both API

IR spectrum indicated the presence of an overtone band

and formed tablets. Particular attention should be paid to

-1

of primary aromatic amine (4455 cm ), aromatic carbon-

773

impurity also obtainable in starting raw materials.

storage temperature of API, sugar or starch excipients


NELSON A ET AL: IMPURITIES RESPONSIBLE FOR CHANGE IN PHYSICAL CHARACTERISTICS and formulated tablets as the rate of formation of o

degradation can however be minimized and probably

glucosamine is increased by 2 – 3 times for each 10 C

prevented if these factors are eliminated. GC-MS and/or

rise in temperature. Preferably the dry granulation

TLC were seen to be relatively simple and readily

method is recommended over wet granulation method

available analytical means to achieve this.

especially for recurrent cases. Most importantly, the quality of starting materials should be known and the impurities in them should not exceed the officially stated limits. Lastly, the use of reducing sugars like lactose as an excipient should be discouraged especially in situations where it is impossible to quickly verify the quality of starting raw materials shortly before compression. The darkening of paracetamol tablets on storage and shortly after compression which was seen and assumed to be a physical degradation was found to be predominantly a chemical degradation favored by moisture, extreme pH, high temperature and the presence

Fig 1: C = Totally degraded paracetamol tablets, E = Partially degraded paracetamol caplets.

of certain impurities in the starting raw materials. This

Fig 2: Gas chromatogram of methanolic extract of darkened paracetamol tablets Peak identification: 2 = Paracetamol, 7 = 4-aminophenol, 8 = N-phenylglucosamine, 9 = N-(ethylphenylether) glucosamine

774


NELSON A ET AL: IMPURITIES RESPONSIBLE FOR CHANGE IN PHYSICAL CHARACTERISTICS

Fig 3: TLC of reference paracetamol (E), darkened Paracetamol (C) and 4-aminophenol (AP) Spot identification: E= paracetamol (RF=0.71), C=darkened paracetamol (RF=0.71, 0.83, 0.91), AP= 4-aminophenol (RF=0.91)

4-aminophenol OH -

Fig 4: Proposed degradation route

775


NELSON A ET AL: IMPURITIES RESPONSIBLE FOR CHANGE IN PHYSICAL CHARACTERISTICS REFERENCES 1)

Roy J. Pharmaceutical impurities-A mini Review. AAPS Pharm. Sci.Tech. 2001;3:2

2)

ICH topic Q3 A . Impurities testing guideline: Impurities in new drug substances, the European agency for the evaluation of medicinal products human medicine evaluation unit.(1995)

3)

Van krimpen PC, Van Bennekom WP, and Bult A. Penicillins

and

cephalosporins:

Physicochemical

properties and analysis in pharmaceutical and biological matrices. Pharm. Weekbl[Sci]. 1987 ;9:123. 4)

Alsante KM, Bovtres P, Coutrier MA, Fridmann RC, Harwood Jw, Horon GJ, Jensen AJ, liu Q, Lohr l, Morris R, Raggon JN, Reid GL, Santafianos DP, Sharp TR,. Tucker JL, Wilcox GE.Pharmaceutical impurity identification: A case study using a multi disciplinary approach. Journal of Pharmaceutical Sciences 2004; 93(9):2296.

5)

Oladimeji

FA,

Iranloye

TA.

Survey

type

of

Analgesic/Antipyretic and Antimalarial drugs used in Nigeria. Pharmacy World Journal 1990; 7:123-128 6)

Ellis and Frank. Paracetamol: a curriculum resource. Cambridge: Royal society of chemistry , 2002

7)

British

Pharmacopoeia

Vol

II.

Her

Majesty’s

stationery office, London ,2008 pp. 1653-1654. 8)

Laurence HM, Christopher MJ .Experimental organic chemistry:

Principles

and

practice.

Blackwell

Publishing,1999 pp 292 9)

Dutta S, Danell A. GC-MS sample preparation guidelines.http://www.ecu.edu (accessed 22/1/10)

10)

Kenyon AS, layloff TP.Compendium of unofficial methods for rapid screening of Pharmaceuticals by thin-layer

chromatography.

Journal

of

AOAC

international.1995; 78(1):41-49. 11)

Charlie scandrett. Maillard Reactions 101: Theory. http.//brewery.org/library/maillardcso497.hmtl(accessed 23/1/10)

776
