Abstract: The spectrophotometric and microbiological assay methods used for the determination of ampicillin levels in ampicillin/cloxacillin combinations were compared. A total of 24 samples of ampicillin/cloxacillin suspensions from 8 batches and 3 companies (B 2 , D and E) were assayed for their ampicillin content levels using the Spectrophotometric assay method. Infra-red (IR) Spectroscopy confirmed the presence of ampicillin in all the samples and the results obtained showed that the samples had a wide range of amounts of ampicillin which compared favourably with the microbiological assay results of the same products. All the products assayed had ampicillin content within acceptable ranges of the BP and USP. Spectrophotometric assay is recommended for the quick determination of the levels of ampicillin in their combinations with cloxacillin

C E Udobi

E I Akpabio

P M E Ubulom

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Current Research Journal of Biological Sciences 3(1): 31-34, 2011
ISSN: 2041-0778
© Maxwell Scientific Organization, 2011
Received: October 16, 2010 Accepted: December 02, 2010 Published: January 15, 2011
Corresponding Author: C.E. Udobi, College of Science and Technology, Kaduna Polytechnic, Kaduna, Nigeria
31
Comparative Studies of the Spectrophotometric and Microbiological Assay
Methods Used for the Determination of Ampicillin Levels in
Ampicillin/Cloxacillin Combinations
1C.E. Udobi, 2P.M.E. Ubulom and 2E.I. Akpabio
1College of Science and Technology, Kaduna Polytechnic, Kaduna
2Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,
University  of Uyo, Nigeria
Abstract: The spectrophotometric and microbiological assay methods used for the determination of ampicillin
levels in ampicillin/cloxacillin combinations were compared. A total of 24 samples of ampicillin/cloxacillin
suspensions from 8 batches and 3 companies (B2, D and E) were assayed for their ampicillin content levels
using the Spectrophotometric assay method. Infra-red (IR) Spectroscopy confirmed the presence of ampicillin
in all the samples and the results obtained showed that the samples had a wide range of amounts of ampicillin
which compared favourably w ith the microbiological assay results of the same products. All the products
assayed had ampicillin content within acceptable ranges of the BP and USP.  Spectrophotometric assay is
recommended for the quick determination of the levels of ampicillin in their combinations with cloxacillin.
Key words: Ampicillin, cloxacillin, Infra red, spectrophotometric assay, spectroscopy
INTRODUCTION
The Spectrophotometer is an analytical photoelectric
instrument which is used for estimating the concentrations
of solutions by measuring either the amount of light
passing through (transmittance) or the amount of light
absorbed (Absorbance). It  is known to work on the
principle of the Beer Lamberts law and within the range
in which the law is obeyed, it has been effectively used in
the assay of pharmaceuticals when made into solutions
(Eboka et al., 1997). The method which involves
principally the use of the spectrophotometer for this
purpose is  termed, spectrophotometric assay.
Spectrophotometric assay of pharmaceuticals is highly
dependent on the chemical reactions of the active
ingredients of the agent concerned e.g., the concentration
of the active ingredients or its characteristic end product
can be determined by comparing its light absorption with
that of a solution of the same substance at the same
standard concentration.
Microbiological assays are generally used in the
determination of the potency of growth inhibiting
substances especially antibiotics (William, 2005). This
involves the use of suitable microorganisms as the
biological system. Microbiological assay methods,
employs the use of the biological properties of medicinal
agents in the estimation of their activity. The method uses
the basic principle of comparing a sample of known
activity or potency (standard) and one of unknown
activity at the same time and under very strict comparable
conditions (W illiam, 2005; Udobi and Onaolapo, 2010).
Ampicillin and Cloxacillin are semi-synthethic
penicillins which share very close similarity in their
structures due to the presence of the Beta lactam and
thiazolidine rings. This explains why the design of the
methods for their selective determination in combined
preparations must be done with a high degree of
selectivity in mind.  Penicillanic and Penicilloic acids, the
acid and base hydrolysis products, respectively of
penicillin have been found to react with formaldehyde to
give a pyrazinone derivative in acidic medium. A
wavelenght of 268 nm, was initially used to determine the
level of ampicillin in the presence of cloxacillin but was
found to be interfered with by readings from cloxacillin
and other additives in pharmaceutical preparations
(Akanni and Ayim, 1992). A wavelength of 373 nm
where there will be no such  interference was therefore
suggested. Presently, the component ampicillin in
combination of ampicillin and cloxacillin can successfully
be determined using microbiological assays. 
This study presents the results of the comparison
between spectrophotometric and microbiological assay
methods for the determination of the levels of the
ampicillin content of combined preparations of
ampicillin/cloxacillin suspensions.
Curr. Res. J. Biol. Sci., 3(1): 31-34, 2011
32
MATERIALS AND METHODS
Materials: The basic materials used were standard
ampicillin powder obtained from National Agency for
Drug Administration and Control (NAFDAC) in Nigeria,
Different ampicillin/cloxacillin suspensions sold within
Kaduna and Zaria environs and Spectrophotometer-(Pye-
unicam) from Faculty of Pharmaceutical sciences,
Ahmadu Bello University, Zaria-Nigeria where the
experiments were carried out. 
Calibration curve for standard ampicillin: A
calibration curve for standard ampicillin was drawn using
results obtained from reactions of ampicillin as described
by Akanni and Ayim, (1992) (at concentrations18 g - 91
:g. A stock solution containing 1  mg/mL of standard
ampicillin was made and 2,4,6,8 and 10 mL aliquots of
the stock were taken into separate flasks and hydrolysed
using 2 mL of 2 M sodium hydroxide. After 20 min, 1 mL
of 2 M sulphuric acid was added to each mixture. 50 mL
of formaldehyde containing phosphate buffer PH 2.5 was
added to each flask and heated for one hour at 100oC.
Each of the flasks so heated was designated solution A.
Equivalent amounts of aliquots (2, 4, 6, 8 and 10 mL) of
the same 1 mg/mL stock solution of standard ampicillin
were taken in another set of flasks. Each sample was
reacted with 50 mL formaldehyde containing phosphate
buffer (PH 2.5). The flasks were each designated solution
B. A 1:2 dilution of corresponding solution A and B were
made with 2 M sulphuric acid and the absorbance of
solution A was measured at the wavelenght 373 nm using
solution B as reference. The absorbance reading so
obtained were used with the corresponding concentrations
to plot a calibration curve for ampicillin.
Determination of ampicillin in ampicillin/cloxacillin
oral preparations:
Spectrophotometric assay: A stock solution of
ampicillin/cloxacillin oral preparations containing 1
mg/mL of ampicillin was made from reconstituted
samples of concentration of 250 mg/mL.  Aliquots (5 mL)
of the stock solution was then used to prepare reaction
mixtures to make solutions A and B as done during the
calibration. A 1:2 dilution of corresponding solutions of
solutions A and B were made using 2M sulphuric acid
and the absorbance of solution  A determined w ith solution
B as reference. This procedure was strictly followed for
all the preparations tested by this method. The absorbance
due to each sample is compared with that due to 5 mL of
the standard ampicillin in the calibration curve.
Microbiological assay: The microbiological assay was
done using the method described by Udobi et al. (1994,
2010) which involves the use of Bacillus megatharium
NCTC 10342A76 AS indicator organism.
Preparation of standard test doses: Three
concentrations of standard ampicillin trihydrate powder
10, 20 and 40 mg/mL were prepared on the day of assay
using sterile 0.1 M phosphate buffer (usp) as diluent
bearing in mind that 1.15 g of pure ampicillin trihydrate
contains 1 g of ampicillin.
Preparation of sample test doses: Samples of ampicillin/
cloxacillin suspensions were first reconstituted according
to the instructions of the manufacturers. Three
concentrations of 10, 20 and 40 mg/mL of each sample
were then prepared on the day of the assay using sterile
0.1M  phosphate buffer (usp) as diluent.
Assay procedure: The agar diffusion method was used.
The 6x6(3x3) dose level latin square design using large
plates was employed. 200 mL of sterile nutrient agar
inoculated with 1 mL of the test culture of OD470 of
0.17(this contains approximately 1 million cells of
Bacillus megatharium NCTC 10342A 76) was poured on
top of the basal layer aseptically and was also allowed to
set. Using a sterile No.4 cork borer, 36 cups were cut in
the agar in a random manner referred to as the latin square
design. 20 :L of each concentration(10, 20 and 40
mg/mL) of the sample and agents were then applied into
each cup as prescribed by the  latin square design method.
The plates were left to stand for two hours to allow for
effective diffusion. They were then incubated at 37ºC for
between 18-24 h. Zones of inhibition produced by both
the standard and test concentrations were measured to the
nearest millimeter. The experiments were duplicated and
the average of the zones of inhibition (ZOH) were taken.
RESULTS
Up to a concentration of 91 :g/mL, the
corresponding OD 373 nm absorbance readings obtained
using the spectrophotometric method showed a linear
relationship with concentration (Fig. 1). This means that
up to this concentration and using the method of Akanni
and Ayim (1992), ampicillin content can still be
determined by absorbance readings. The results agree
with the method and goes further to show that even at this
high concentration (up to 91 :g/mL), a linear relationship
between absorbance and concentration could still be
obtained. (Table 1).
Table1: Absorbance reading s of the c once ntrations o f standa rd
ampicillin (18-91 :g/mL)
Volume of stand ard Concentration (:g/mL) Absorbance  at 373 nm
2 18 .2 0.4
4 36 .4 0.8
6 54 .6 1.4
8 72 .8 1.9
10 91 .0 2.4
Curr. Res. J. Biol. Sci., 3(1): 31-34, 2011
33
Table 2: Re sults for the Spectrophotom etric assay of ampicillin in ampicillin/cloxacillin combinations from co mpanies B 2,  D and E
B 2 D E
--------------------------------------------------- ---------------------------------------------------- ---------------------------------------------------
Calculated Corresponding Calculated Corresponding Calculated Corresponding
Comp anies Absorbance p ote nc y (% ) concentration Absorbance p ote nc y (% ) concentration Absorbance p ote nc y  (% ) concentration
Batch 1
1 1.20 104.3 130.37 1.10 95.60 119.50 1.03 89.50 111.87
2 1.18 102.6 128.25 1.20 104.3 130.37 1.04 90.49 113.11
3 1.20 104.3 130.37 1.10 95.60 119.50 1.08 93.91 117.38
Batch 2
1 1.18 102.6 128.25 1.06 92.17 115.21 1.28 113.0 141.25
2 1.14 99 .0 123.75 1.10 95.60 119.50 1.30 111.0 138.87
3 1.14 99 .0 123.75 1.08 93.90 117.37 1.30 113.0 141.27
Batch 3
1 1.28 113.3 139.12 1.16 100.8 126.00
2 1.28 113.3 139.12 1.14 99.13 123.91
3 1.28 113.3 139.12 1.19 103.4 129.25
Table 3: Lev els of ampicillin in ampicill in/cloxacillin combinations (expressed in %  age) from com panies B 2, D and E determined using
microbiological method
B 2 D E
------------------------------------------------- --------------------------------------------------- ------------------------------------------------------
Calculated Equivalent Calculated Equivalent Calculated Equivalent
Comp anies p ote nc y (% ) concentration :g/5 mL p ote nc y (% ) concentration :g/5 mL p ote nc y  (% ) concentration
Batch 1
1 104.80 131.00 89.20 111.50 91.80 114.75
2 97.80 122.25 91.16 113.95 92.50 115.62
3 102.40 128.00 90.15 112.68 90.20 112.75
Batch 2
1 90.80 113.50 89.50 111.80 98.80 123.50
2 89.10 111.37 90.20 112.75 107.50 134.37
3 92.90 116.12 89.12 111.40 109.20 136.50
Batch 3
1 112.20 140.25 92.60 115.75
2 109.80 137.25 95.71 119.63
3 108.00 135.00 103.10 128.75
Fig. 1: Calibration curve for ampicillin at 373 nm, the
calibration curve gave a straight line of reproducible
linearity
DISCUSSION
Pharmaceutical quality checks is an important aspect
of its manufacture which no manufacturer is willing to
compromise for obvious reasons. For these same reasons,
no consumer will knowingly accept a compromise on the
quality of drugs he or she takes (U dobi et al., 1994).
The combination of ampicillin and cloxacillin is
known to have a synergistic effect (Ayim et al., 1990). Its
mode of action has also been suggested to be by a
combination effect especially when penicillinase
producing   organisms   are   involved   (Akanni  and
Ayim, 1992). Here, cloxacillin is believed to block the
production of penicillinase while the ampicillin goes to
destroy the organism. Whatever the method, the fact
remains that the combination as a dosage form will not
have any desired effect when either of the combinants is
below the standard recommended level. The synergy
between the two penicillins, makes the determination of
the level of one in the combination ted ious especially
when using the age long and reliable microbiological
method.
The result of all the samples assayed using the
spectrophotometric method showed that they all contained
acceptable levels of ampicillin in the combination by USP
and BP standards (Table 2). The same samples were also
assayed using the microbiological method and the results
obtained are showed the same (Table 3). The highest
difference in the calculated percentage potency of
Curr. Res. J. Biol. Sci., 3(1): 31-34, 2011
34
different bottles of the same batch using the
spectrophotomertric method, is that obtained from the first
batch of the products of company D which is 9% and that
obtained from the 2nd batch of company E using the
microbiological assay method which is also 9%. Other
batches showed differences within 0-7%. If these errors
are either from the weighing, mixing or other
manufacturing process, they are within reasonably
accepted ranges. The spectrophotometric method as
described by Eboka et al. (1997) can therefore, be said to
be very reliable for the determination of ampicillin levels
in its combination with cloxacillin due to its specificity
among others. It can very well be applied in ampicillin
determination up to a concentration of 91 :g/mL (Fig. 1)
which covers the recommended  dosage of ampicillin in
its combination with cloxacillin  and compares very
favourably with the microbiological assay method using
Bacillus megatharium NCTC 10342A 76 as indicator
organism as described by Udobi et al. ( 1994).
CONCLUSION
The spectrophotometric assay method used is not
tedious and takes less time when compared with the
microbiological method. Generally, the two methods
compare favourably. One problem that may be
encountered with the spectrophotometric method
however, is its high specificity. This is because, a little
change in absorbance reading which can result from any
mistake in timing or weighing will make an appreciable
difference in the corresponding percentage potency. It
requires a very high degree of accuracy in all the steps
and very skilled personnel if reproducible results are to be
obtained. However, the simplicity, straight forward nature
and  the  short time this method takes can be exploited at
entry posts for on the spot checks of the levels of
ampicillin content in this kind of combinations entering a
country or a region.
ACKNOWLEDGMENT
We want to thank ‘Oga’ Tanko of the Microbiology
Laboratory of the Faculty of Pharmaceutical sciences A B
U zaria for his immense assistance. 
REFERENCES
Akanni, A.O. and J.S. Ayim, 1992. Determination of
ampicillin in the presence of cloxacillin. J. Pharm.
Biomed Anal., 10(1): 43-47.
Ayim, J.S.K., A.O. Akanni and T.O. Dawodu, 1990.
Quality of ampicillin/cloxacillin  preparations in the
Nigerian market.  Nig. J. Pharm., 20: 36-38.
Eboka, C.J., S.O. Aigbavboa and J.O. Akerele, 1997.
Colometric determination of the fluoroquinolones. J.
Antimicrob. Chemother., 39: 639-641. 
Udobi, C.E., J.A. Onaolapo and P.F. Olurinola, 1994.
Determination of ampicillin in ampicillin/cloxacillin
suspensions marketed in kaduna and zaria environs.
West Afr. J. Pharm., 8(2): 55-60.
Udobi, C.E. and J.A. Onaolapo, 2010. Microbiological
assay of the active component of ampicillin in
ampicillin and ampicillin/cloxacillinsuspensions
using Bacillus megatharium NCTC 10342A76 as
indicator  organism.  Afr.  J.  Microbiol. Res., 4(1):
51-54.
William, H., 2005. Microbiological Assay for
Pharmaceutical Analysis: A Rational Approach.
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Comparative Studies of the Spectrophotometric and Microbiological Assay Methods Used for the Determination of Ampicillin Levels in Ampicillin/Cloxacillin Combinations

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Comparative Studies of the Spectrophotometric and Microbiological Assay Methods Used for the Determination of Ampicillin Levels in Ampicillin/Cloxacillin Combinations

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