American Journal of Anal yt ical Chemistry, 2011, 2, 491-499
doi:10.4236/ajac.2011.24059 Published Online August 2011 (http://www.SciRP.org/journal/ajac)
Sequential Injection Analysis of Ampicillin in
Pharmaceuticals by Using Potentiometric Detectors Based
on PVC and Sol-Gel Membranes
Nestor Zárate1, Alberto N. Araujo2, M. Conceiçao B. S. M. Montenegro2, Ricardo Pérez-Olmos1*
1Dpto. Química Analítica, Escuela Universitaria de Ingeniería Técnica Industrial,
Universidad del País Vasco, Plaza de la Casilla 3, 48012, Bilbao,
2REQUIMTE, Dpto. de Química Física, Faculdade de Farmacia,
Universidade do Porto, Rua Anibal Cunha 164, 4050 Porto, Portugal
Received April 1, 2011; revised May 12, 2011; accepted May 26, 2011
Abstract
This work describes the construction and the evaluation of the general performance of new ampicilli-
nate-selective electrodes based on manganese (III) tetraphenylporphyrin [Mn(III) TPP-Cl] as ionophore, in-
corporated in both PVC and sol-gel membranes and directly applied onto a conductive graphite/epoxy resin
support. The units were constructed without inner reference solution adopting conventional configuration
and in the case of PVC membrane the tubular configuration was also adopted. The good working character-
istics of these electrodes, made possible its application to the determination of ampicillin in pharmaceuticals
formulations, both in conventional batch analysis and in flow conditions, when the electrodes were coupled
to a SIA system. In the last case the potentiometric sensors presented linear response towards ampicillin
concentration between 5.0 × 10–4 and 5.0 × 10–2 mol·l–1 with slopes of –57.4 and –63.5 mV·dec–1 for the PVC
and sol-gel membranes, respectively. The developed procedures enable mean relative standard deviations
better than 3% for all the samples analysed. The obtained results do not statistically differ from those fur-
nished by applying the HPLC reference method.
Keywords: Ampicillin, SIA, Potentiometric Detection, Pharmaceutical Formulations
1. Introduction
Several PVC membranes based on metalloporphyrins
have been used on the construction of selective elec-
trodes for the potentiometric determination of organic
species with pharmaceutical interest such us: salicylate
[1], penicillin-G [2], valproate [3,4], ibuprofen [5],
2-hydroxybenzyhydroxamate [6] and diclofenac [7]. The
use of plasticized PVC membranes for electrodes con-
struction has the disadvantage of the gradual leaching of
the plasticizer and the electroactive components from the
membrane into the sample solution. This fact deteriorates
some sensor characteristics like slope and signal-to-noise
ratio, thus limiting the durability of these membranes [8],
being this effect more important when the electrode is
used in flow conditions. Siloxane polymers are very
promising materials for the development of ion-selective
membranes by the way of sol-gel technology which have
permitted to design hybrid inorganic-organic materials,
giving an access to the fabrication of glasses and films of
network structure with a controlled porosity and thick-
ness which showed high physical and chemical stability
[9]. Sol-gel process has been employed to prepare ISEs
selective to chloride [10,11] and copper [12] by using
tridodecylmethylammonium chloride and thiosemicar-
bazone, respectively, as ionophores. Recently, a val-
proate-selective electrode based on Mn (III)-tetrapheny-
lporphyrin incorporated in a sol-gel matrix was previ-
ously reported [4]. Some of these sol-gel sensor mem-
branes were used as potentiometric detectors in FIA [11]
and SIA [4] systems.
Ampicillin is one of the most important antibiotics
since exerts an effective bactericidal action against both
Gram-positive and Gram-negative organisms. Potenti-
ometric determination of ampicillin has been initially
carried out by using ISEs sensitive to ampicillinate with

N. ZÁRATE ET AL. 492
membranes based on different ionophores such as: qua-
ternary ammonium salts [13,14], ampicillin-phosphotun-
gstate ion pair [15] and azacompounds and metal phta-
locyanines [16]. The use of penicillinase have been pro-
posed to construct enzyme electrodes which were applied
to the determination of various
 -lactamic antibiotics
[17,18]. The use of conventional and enzyme potenti-
ometric detectors in pharmaceutical analysis has been
receantly reviewed [19,20].
In this work, a novel ampicillinate selective electrode
has been constructed and evaluated using the metal-
loporphyrin [Mn (III)-TPP-Cl]. The solid-contact elec-
trodes were prepared both with PVC and sol-gel mem-
branes, with conventional configuration. In the case of
PVC membrane a tubular configuration was also per-
formed. The constructed detectors were coupled to a se-
quential injection analysis (SIA) system and applied to
the analytical control of ampicillin in pharmaceutical
formulations (injectables).
2. Experimental
2.1. Reagents and Solutions
The sensor membrane for both PVC and sol-gel elec-
trodes were prepared using 5,10,15,20 tetrapehenylpor-
phirinate manganese (III) (Mn(III)TPP-Cl) as ionophore.
The PVC membrane electrodes consist in a mixture of
o-nitrophenyloctylether (o-NPOE) as plasticizer, and
high molecular weight PVC as inert matrix, previously
dissolved in tetrahydrofuran. The sol-gel membrane elec-
trodes were prepared using methyltrietoxysilane (MTES),
as silanol precursor, ethanol, hydrochloric acid and poly-
ethyleneglycol (PEG 6000), for avoiding a fast mem-
brane drying, and deionized water. The conductive sup-
port, in both electrodes, was a mixture (1:1.2 w/w) of
graphite powder and a non-conductive epoxy resin (Ar-
aldite M and hardener HY) respectively.
For the PVC electrodes, the stock solution used to
prepare the calibration curves in the potentiometric de-
terminations was a 1.0 × 10–1 mol·l–1 sodium ampicillin
prepared in a 5.0 × 10–2 mol·l–1 dihydrogenphosphate
solution (pH 4.5). In the case of the sol-gel electrodes,
the sodium ampicillin stock solution was dissolved in a
1.0 × 10–2 mol·l–1 morpholinoethanesulfonic acid solu-
tion (pH 3.8). These ampicillin stock solutions were pre-
pared daily because of its instability.
The 5.0 × 10–2 mol·l–1 dihydrogenphosphate solution
was used for the ionic strength and pH adjustment and
also as carrier solution in the potentiometric determina-
tion with PVC membrane electrodes in flow conditions.
Besides the outer chamber of the double junction refer-
ence electrode was filled up with the above mentioned
solution. In the case of the sol-gel membrane electrodes
the 1.0 × 10–2 morpholinoethanesulfonic acid solution
was used for the ionic strength and pH adjustment, as
carrier solution and also for filling up the outer chamber
of the double junction reference electrode. Both ISA/
carrier solutions contained 1.0 × 10–8 mol·l–1 sodium am-
picillin for the rapid reestablishment of the potentiomet-
ric baseline.
For the chromatographic assays the mobile phase A
consists in: 0.5 ml of diluted acetic acid solution (1.2 g of
glacial acetic acid in 10 ml of deionized water), 50 ml of
a 2.0 × 10–1 mol·l–1 dihydrogenphosphate solution and
50 ml of acetonitrile was made up to 1000 ml with de-
ionized water. Mobile phase B was prepared in the same
way than mobile phase A but in this occasion 400 ml of
acetonitrile were added. Both mobile phases were de-
gasified and filtered through a Millipore 0.45 µm filter.
All solutions were prepared with distilled and deion-
ized water with conductivity less than 0.1 µS·cm–1, and
analytical grade reagents were used without any addi-
tional purification.
2.2. Apparatus
Potentiometric measurements were carried out by using a
Crison 2002 potentiometer (sensitivity  0.1 mV) cou-
pled to an electrode switcher, of the same brand, that
recorded the potential difference between an Orion Re-
search 92-02-00 double junction reference electrode and
the corresponding indicator electrode. To determine the
pH value of all solutions an Orion Research 91-02-00
combined glass electrode was used.
In constructing the SIA system (Figure 1), a Gilson
Minipulse 3 four-channel peristaltic pump was used to
ensure flow of the solutions. A CheminertTM C25-3
188D multiposition rotatory valve of 8 ports, electrically
actuated, was used to select an adequate solution. With
the purpose of guaranteeing the maximum reproducibil-
ity of the aspirated and forward volumes, an NResearch
161 T031 three-way solenoid valve (SV) was placed
between the peristaltic pump and the selection valve. All
of the SIA system components were connected with
PTFE tubes (i.d. 0.8 mm). The reaction coil was 50 cm in
length and the holding coil was 200 cm and was coiled
over a plastic net. The selective and the reference elec-
trodes were fitted in home-made plastic supports con-
structed as described elsewhere [21] with slight modifi-
cations. With the aim of eliminating electric noise, the
constructed electrodes were connected to a home-made
ground electrode [22]. For the data adquisition a single
channel Kipp&Zonen BD11 stripchart recorder was used.
All the SIA system operations were controlled by a com-
puter through an Advantech

N. ZÁRATE ET AL. 493
Rec
mV
Figure 1. Schematic representation of the potentiometric SIA system developed. P: per istaltic pump; RV: Multiposition rota-
tory valve; SV: Three-way solenoid valve; HC: Holding coil; RC: reaction coil; GE: Ground electrode; D: PVC or sol-gel
membrane.
PCL-711S interface card by running software written in
Quick Basic 4.5 programming language.
The liquid chromatography equipment used for the
ampicillin determinations was a model Hewlett-Packard
(1050 series) with a Hewlett-Packard (1040M series II
DAD) as detector, and a Spherisorb ODS2 (250 × 4 mm),
5 µm particle size, column from Teknochroma. For the
equipment control and data acquisition a ChemStation,
from Agilent Technologies, was used.
2.3. Construction of the Electrodes
The PVC membrane for the ampicillin selective elec-
trodes, of conventional configuration, were prepared as
previously reported [23] by using Mn(III)TPP-Cl (1 wt%)
as an ionophore, o-NPOE (66 wt%) as plasticizer solvent
and PVC (33 wt%), previously dissolved in tetrahydro-
furan, as polymeric matrix. The membranes were applied
dropwise over the graphite conductive support and dried
at room temperature overnight. The tubular PVC detec-
tors were constructed as described elsewhere [24] using
the same membrane composition.
The sol-gel membranes were prepared as previously
reported [4], with slight modifications, by mixing 4.5 ml
of alcoxy reagent (MTES), 1.5 ml double-deionized wa-
ter, 1.0 ml ethanol and 100 µl of a 1.0 × 10–1 mol·l–1 hy-
drochloric acid solution, in a 50.0 ml PTFE beaker. After
well-mixing, 25.0 mg of Mn(III)TPP-Cl was added to the
solution followed by 50 µl of PEG 6000 which avoided
fast membrane drying and consequent brittleness. This
mixture was left overnight under stirring until the sol-gel
solution was achieved and then the conductive graphite
surface was immersed into the viscous solution for 5 s.
The obtained membranes were dried at 20ºC for five
days.
2.4. Procedures
2.4.1. Sampl e Preparation
Pharmaceutical formulation samples (injectables), con-
taining sodium ampicillin, were prepared by rigorous
weighing out of 200.0 mg of the solid and subsequent
dissolution in 100.0 ml of a 5.0 × 10–2 mol·l–1 dihydrogen
phosphate or 1.0 × 10–2 mol·l–1 morpholinoethanesulfo-
nic acid (HMES) solutions when the PVC membrane or
the sol-gel membrane electrodes were respectively used.
All the samples were prepared to have a final ampicillin
concentration that fitted in the linear concentration range
of the developed electrodes.
2.4.2. De ve l op e d Batch Procedure
An aliquot of 50.0 ml, of the prepared ampicillin sample,
was pipetted directly into a 150 ml beaker and the PVC
or sol-gel membrane electrodes and the reference elec-
trode were immersed in the magnetic stirred solution.
Finally, the sample concentration was determined by
means of direct potentiometry using calibration curves
prepared with ampicillin concentration ranging from 1.0
× 10–4 to 1.0 × 10–2 mol·l–1.
2.4.3. Developed SIA Procedure
Once the SIA system had been set up, all the PTFE tubes
were filled with the carrier/ISA solution placing the peri-
staltic pump in the propulsion mode. This operation
mode was maintained until a stable baseline for the po-
tentiometric detector was obtained. The ampicillin solu-
RV
HC
SV
Ampicillin stock
solution / Sample
1
RC
GE Waste
R
P
ISA
D
PVC ¯ SG

N. ZÁRATE ET AL. 494
tions, of the calibration curve, 5.0 × 10–4, 1.0 × 10–3, 5.0
× 10–3, 1.0 × 10–2 and 5.0 × 10–2 mol·l–1 were prepared
from the 1.0 × 10–1 mol·l–1 sodium ampicillin stock solu-
tion. Only two steps were required for getting the ana-
lytical signal and they were applied to each solution.
When the PVC membrane electrodes were used, 165 µl
of ampicillin stock solution were aspirated through port 5
of the rotatory valve at a flow of 1.3 ml·min–1 and sent to
the detector through port 6 at a flow rate of 1.5 ml·min–1.
However, when the sol-gel membrane electrodes were
employed, 250 µl of ampicillin stock solution were aspi-
rated and sent to the detector at the aforementioned flow
rates. The pharmaceutical samples suffered the same
procedure.
2.4.4. Reference Procedure
The HPLC method used for the determination of am-
picillin in the pharmaceutical samples was similar to the
chromatographic method proposed by the European
Pharmacopoeia [25]. Therefore, 30.0 mg of solid sample
were rigorous weighed and dissolved in 10 ml of mobile
phase (A/B), formed by 75% of mobile phase A and 25%
of mobile phase B. From this solution, 0.1 ml were ex-
actly pipetted and transferred to a 25.0 ml volumetric
flask and diluted with the mobile phase. Afterwards, an
aliquot of 20 l from this solution was injected into the
HPLC equipment. The separation and detection were
carried out according to the following chromatographic
conditions: elution mode, isocratic; flow rate, 1.0 ml
min-1; detection wavelength, 254 nm; temperature, am-
bient; retention time expected, 3.32 min; analysis time,
5.5 min.
3. Results and Discussion
3.1. Working Characteristics of Electrodes with
Conventional Configuration
In order to determine the influence of the immobilizing
support on the performance of the electrodes selective to
ampicillinate, the metalloporphyrin Mn(III)-TPP-Cl was
incorporated both into PVC or ceramic (sol-gel) mem-
branes. The working characteristics of both types of
conventional electrodes constructed were evaluated on
the basis of the IUPAC recommendations [26]. The ex-
periments were performed in solutions with the ionic
strength adjusted with the 5.0 × 10–2 mol·l–1 dihydro-
genphosphate solution when the PVC membranes were
evaluated or with the 1.0 × 10–2 mol·l–1 HMES solution
in the case of sol-gel membranes. From the data, pre-
sented in Table 1, it is possible to affirm that both types
of ampicillinate selective electrodes constructed in this
work, using the same ionophore, presented similar or
better slopes, reproducibilities, response times and prac-
tical limits of detection than those reported by other au-
thors [13-16]. In spite of having better reproducibility
and similar response time, the sol-gel membrane elec-
trode showed narrower linear concentration range and
shorter life time when compared with that obtained with
PVC membrane electrode.
The Reilley diagrams obtained for the PVC membrane
electrode evidenced an operational pH range between 3.5
and 8.0 for the higher concentration of ampicillinate (1.0
× 10–2 mol·l–1) tested; however, for lower concentration
of ampicillinate (1.0 × 10–4 mol·l–1), the operational pH
range clearly diminishes, mainly in the basic zone. This
is probably due to the fact that with an increase of OH-
relatively to ampicillinate, the response towards this ion
overlaps the response towards the primary ion. Similar
behaviours has been previously described when PVC
membranes electrodes selective to other organic com-
pounds, using the same metallporphyrin, were con-
structed and evaluated by some of us [2,4,27]. For that
reason the working pH chosen for all measurement con-
dition was 4.5 which guaranteed, according to the peni-
cillin pKa (2.5) the complete dissociation of the analyte.
In the case of sol-gel membrane electrodes, the Reilley
diagrams showed that the potential measurements of the
electrode changed according to the pH of the ampicilli-
nate solution. This fact previously reported [28], was due
to the presence of silanol groups (Si-OH) at the surface
of the ceramic membrane which makes these materials
sensitive to pH changes. So, when this type of electrode
was used, a strict control of sample pH (3.8) was needed.
Interference caused by some common inorganic ani-
ons was evaluated by carrying out the determination of
the selectivity potentiometric coefficients (log Kpot) by
the separate solution method at pH 4.5 and 3.8 for the
PVC and sol-gel membranes respectively, and in the
concentration range from 1.0 × 10–4 to 1.0 × 10–2 mol·l–1
for both primary and interfering ions. The results showed
in Table 1 were obtained after two determinations per-
formed with three electrodes and reveal that the devel-
oped electrodes were fairly selective to ampicillinate
over other anions, being their selectivity coefficients
similar or better than those obtained by other authors
[13,16]. Regarding the Hofmeister selectivity pattern of
the classical liquid ion-exchanger, the developed PVC
and sol-gel membranes exhibited different selectivity
sequences from that one as previously demonstrated
when the same metalloporphyrin had been used to con-
struct other anion selective membrane electrodes, namely
carboxylic ions [2,4,27]. For PVC membrane electrode
the sequence SCN– > Sal– > I– > NO3– > NO2– > Cl– >
SO42– > H2PO4– > HCO3– was obtained. Regarding the
sol-gel membrane electrode the sequence HCO3– > NO2– >
NO3– > ≈ SCN– > Sal– > I– > Cl– > H2PO4– > SO42– was

N. ZÁRATE ET AL.495
Table 1. General working characteristics of the developed ampicillinate selective electrodes.
Characteristics PVC membrane Sol-gel membrane
Linear concentration range (mol·l–1) 1.7 × 10–5 - 1.0 × 10–1 1.5 × 10–4 - 1.0 × 10–1
Practical limit of detection (mol·l–1) 1.0 × 10–4 1.0 × 10–3
Slope (mV·decade–1) 60.2  0.8 65.3  0.3
Potential stability (mV·day–1)  1.5  0.7
Response time (seconds) < 10 < 10
Lifetime (months) 10 1
Working pH rangea 3.0 - 7.5 -
PVC membrane Sol-gel membrane
Potentiometric selectivity coeffi-
cients a (log Kpot)
Sulphate –1.37  0.02 –1.38  0.06
Dihydrogenphosphate –1.40  0.03 –1.32  0.05
Hydrogencarbonate –1.65  0.02 0.04  0.04
Chloride –0.41  0.01 –0.65  0.05
Nitrate –0.43  0.03 –0.42  0.14
Nitrite –0.22  0.01 0.12  0.08
Iodide 0.68  0.04 –0.45  0.03
Thiocyanate 1.62  0.07 –0.17  0.07
Salicylate 1.45  0.06 –0.22  0.27
a. Ampicillinate concentration = 1.0 × 10–3 mol·l–1
obtained. Being much more hydrophilic in nature, the
sol-gel membrane was less affected by lypophilic anions
such as SCN– which confirm the notion that interferent
action can be reduced by eliminating the plasticizer in
the polymeric membrane [29]. On the other hand, the
fact that the interferent action of the salicilate diminished
when the sol-gel membrane electrode is used, could led
to eliminate significant error for measurement of am-
picillin in biological fluid samples from patients who
have ingested aspirin.
3.2. Behaviour of the Electrodes in the SIA
System
Both type of constructed electrodes were evaluated when
they were coupled in the SIA system depicted in Figure
1 using a 5.0 × 10–2 mol·l–1 dihydrogenphosphate and a
1.0 × 10–2 mol·l–1 HMES solutions as carriers for elec-
trodes with PVC or sol-gel membranes, respectively.
Initially, the aspiration and propulsion flow rates were
estimated by interpolation in graphs obtained by plotting
ml·min–1 vs. revolution per minute (rpm) of the peristal-
tic pump. The intensity and repeatability of the potenti-
ometric signals obtained were studied either by varying
the injection volume of the ampicillinate solution in the
interval 115 - 450 l, and the propulsion rate in the in-
terval 1.2 - 3.5 ml·min–1 in a way, whereby the sampling
rate was not diminished. With the increase of the injec-
tion volume, an increase in the magnitude of analytical
signals was verified up to volumes of 165 and 250 l for
PVC and sol-gel membrane electrodes respectively. For
greater injection volumes no significant improvement in
the signals magnitude was observed requiring more time
and greater consumption of reagents. It was also ob-
served that volumes smaller than 165 and 250 l pro-
vided higher dispersion of the sample zone and conse-
quently lack of linearity. These injection volumes were
obtained for a maximum flow rate of 1.5 ml·min–1 for
both potentiometric detectors. The increments in the flow
rate carried an appreciable loss of signal repeatability,
possibly caused by inertia to the flow during the aspira-
tion stage, and by the dynamic response characteristics of
each electrode type used [30].
The general working characteristics of both electrodes
were evaluated by means of calibration curves obtained
for a range between 1.0 × 10–6 and 1.0 × 10–1 mol·l–1 and
they appear reflected in Table 2. From these data, it is
possible to state that sol-gel membrane electrode showed
narrower linear concentration range and lower life-time
than PVC membrane electrode, but the slope resulted to
be higher. When compared the behaviour of these units
with the results obtained in batch conditions, a small nar-

N. ZÁRATE ET AL. 496
Table 2. Figures of merit of the developed potentiome tr ic SIA system.
PVC membrane Sol-gel membrane
Linear concentration range (mol·l–1) 7.0 × 10–5 – 1.0 × 10–1 7.5 × 10–4 – 1.0 × 10–1
Practical limit of detection (mol·l–1)b 4.0 × 10–5 4.1 × 10–4
Regression equationa E = –57.4 log[C] + 20.1 E = –63.5 log [C] + 19.3
Quadratic correlation coefficient (R2) 0.9992 0.997
Electrode slope and standard deviation
(mV·decade–1) –57.4  0.5 –63.5  0.5
Relative standard deviation (%)c 0.2 0.3
Sampling rate (sample h–1) 40 33
a E: potentiometric signal (mV); log [C]: logarithm of analyte concentration (mol l-1); b Determined according to IUPAC recommendations
[26]; c Obtained for 11 replicates at 1.0 x 10-3 mol l-1 analyte standard solutions
rowing in the linear concentration ranges, an increase in
the practical limit of detection and a diminution of the
slopes were observed with both type of electrodes.
3.3. Analytical Applications
The potentiometric analysis of five samples of pharma-
ceutical formulation (injectables) containing sodium am-
picillin, commercialized in Europe, was carried out with
conventionally or tubular (PVC membranes) shaped ele-
ctrodes according to the batch and SIA procedures. For
comparison purposes the samples were simultaneously
analyzed by applying the HPLC reference method and
the obtained results are summarized in Tables 3 and 4.
The precisions of the developed and the reference me-
thods were evaluated by their simultaneous application to
6 different samples of the same pharmaceutical product
and were expressed in terms of their relative standard
deviations. From the obtained data, it was possible to
affirm that the precisions of both, batch and SIA, poten-
tiometric determinations were good since the mean rela-
tive standard deviation were 1.7% (PVC membrane elec-
trode) and 1.2% (sol-gel membrane electrode) in the SIA
system and 2.1 and 1.6% respectively in batch conditions.
To test whether the developed (batch and SIA) and the
reference methods differed in their precision, a signifi-
cance (two-tailed) F-test was carried out. Since the cal-
culated F values were (just with one exception) less than
the critical F value (7.15), there was no statistically sig-
nificant difference between the standard deviations at
95% confidence level [31]. After three additions of kn-
own concentration of standard sodium ampicillin, the
samples were analysed following the same methods and
the percentages of spike recovery calculated were used to
evaluate the accuracy. No significant differences were
found between the obtained results by applying batch or
SIA procedures. Moreover, slight better results were
founded when sol-gel membrane electrode, instead of
PVC membrane electrode, was used.
On the other hand, when the ampicillin commercial
samples were simultaneously analyzed by applying the
developed potentiometric, batch and SIA, and the HPLC
reference method, a very slight tendency to get lower
values with the potentiometric methods was observed,
since the mean percentage of difference were –1.3% for
the PVC membrane electrode and –1.1% for the sol-gel
membrane electrode in batch conditions and –0.8% for
both type of membrane electrodes when used as poten-
tiometric detectors in the SIA system. In order to estab-
lish whether the developed potentiometric methods could
be accepted as giving reliable results, a two-tailed t-test
with multiple sample (paired by differences) was per-
formed at a P = 0.05 confidence level. The t-data paired
test was applied using the formula: tcalc. = xD
X
S
n,
where xD is the mean difference between the methods
and Sx is the standard deviation of the differences. When
substituting for tcalc., they were found to be –0.62 (PVC
membrane electrode) and –1.11 (sol-gel membrane elec-
trode) in the flow system, and –0.79 and –1.19 respec-
tively in batch analysis. Since all these values were less
than the tabulated t-value (2.78) there was no statistically
significant difference between both potentiometric de-
terminations and the HPLC reference method [31].
The limits of detection of the developed potentiometric
procedures were established according to the Analytical
Methods Committee [32] and following the criteria (
x
+
3 blank). The calculated values resulted to be 80.5 mg·l–1
for the PVC membrane electrode and 88.2 mg·l–1 for the
sol-gel membrane electrode when used as potentiometric
detectors in flow analysis, and 71.7 and 83.8 mg·l–1 re-
spectively in batch conditions. The limits of quantification
(
x
+ 10  blank) were found to be 81.7 mg·l–1 for the
PVC membrane electrode and 90.1 mg·l–1 for the sol-gel
membrane electrode when used in the SIA system, and
79.8 and 93.7 mg·l–1 respectively in batch analysis.
4. Conclusions
The study undertaken has demonstrated that the use of
Mn(III) TPP-Cl as an ionophore for the preparation of

N. ZÁRATE ET AL.497
Table 3. Ampicillinate determination in injectables by simultaneous application of the developed SIA and reference HPLC
methods.
Reference HPLC
method Developed SIA method
PVC membrane Sol-gel membrane
Sample Xa RSDb Xa RSDbRc REd Fe Xa RSDb R
c REd F e
Gobemicina 946.4 ±
12.2 1.3 972.9 ± 21.4 2.2 98.14.6 3.08 971.7 ± 10.81.1 99.3 2.6 1.27
Britapen 1028.4 ±
8.6 0.8 997.5 ± 12.9 1.3 99.5–3.02.25 1020.7 ± 18.81.8 101.2 –0.7 4.78
Hiperbiótico 981.7 ± 7.0 0.7 1008.9 ± 10.2 1.0 102.72.8 2.12 962.7 ± 6.70.7 103.6 –1.9 1.09
Unacim 1050.2 ±
7.9 0.7 1023.9 ± 18.0 1.8 100.3–2.55.19 1031.2 ± 11.61.1 100.8 –1.8 2.15
Amplital 1034.8 ±
9.2 0.9 998.6 ± 21.0 2.1 98.2–3.55.21 1010.6 ± 15.41.5 101.0 –2.3 2.80
aMean ampicillinate concentration and standard deviation (mg/ dose); bRelative standard deviation (%); cMean spike recovery (%); dRelative error of the de-
veloped SIA method versus the reference method (%); eThe critical F value, considering a 95% confidence level and 5 degrees of freedom, for a two-tailed test
is 7.15.
Table 4. Ampicillinate determination in injectables by simultaneous application of the developed batch and reference HPLC
methods.
Reference HPLC
method Batch method
PVC membrane Sol-gel membrane
Sample Xa RSDb Xa RSDbRc REdFe Xa RSDb R
c REdFe
Gobemicina 946.4  12.2 1.3 963.6  11.81.2 97.11.81.07 939.4  10.8 1.1 98.9 –0.71.28
Britapen 1028.4  8.6 0.8
990.3  20.82.1 98.6–3.75.84 1008.9  14.1 1.4 101.7 –1.9 2.70
Hiperbiótico 981.7  7.0 0.7
1014.1  17.21.7 100.23.36.06 1010.2  13.1 1.3 103.1 2.93.50
Unacim 1050.2  7.9 0.7 1005.1  29.42.8 97.7–4.313.85 1011.3  20.2 2.0 100.9 –3.7 6.55
Amplital 1034.8  9.2 0.9
998.6  25.02.5 98.3–3.57.38 1011.0  22.2 2.2 103.8 –2.3 5.84
aMean ampicillinate concentration and standard deviation (mg/ dose); bRelative standard deviation (%); cMean spike recovery (%); dRelative error of the de-
veloped SIA method versus the reference method (%); eThe critical F value, considering a 95% confidence level and 5 degrees of freedom, for a two-tailed test
is 7.15
PVC and sol-gel membranes can give rise to electrodes
with good working characteristics for ampicillinate anion,
which enables the development of a potentiometric me-
thod for its determination in pharmaceutical formulations.
The quality of the obtained results, by using the devel-
oped potentiometric methods, is similar to those fur-
nished by applying the HPLC reference method, being
the attained results slightly better when the sol-gel mem-
brane electrode is used.
On the other hand, the fact that the electrodes can be
easily constructed and incorporated in a SIA system,
showing good performances, offers some advantages
respectively to the batch determination such us: the sys-
tem is easy to operate, the solution handling operations
are reduced, and the proposed method is less time con-
suming with a significant reduction of reagents con-
sumption and waste generation. Besides, the instrumen-
tation is cheaper when compared with the cost of the
equipment required in the HPLC reference method,
which justify the use of potentiometry as an alternative
analytical technique for the determination of ampicillin
in pharmaceutical products.
5. Acknowledgements
This work was supported by the University of the Basque

N. ZÁRATE ET AL. 498
Country (Spain) through Project UPV 171.363-E-15923/
2004. One of the authors (N.Z.) wishes to thank to this
University for the Ph.D. Grant 98/13828/01. We also
thank to Laboratorios Normon S.A, Madrid (Spain), for
samples kindly provided.
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