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Development and validation of HPLC method for determination 
of Ofloxacin and Lomefloxacin in human plasma
Dragica Zendelovska
1
, Trajce Stafilov
2
1
Institute of Preclinical and Clinical Pharmacology and Toxicology, Medical Faculty, 
Sts. Cyril and Methodius University, 50 Divizija bb, 1000 Skopje, Republic of Macedonia; 
2
Institute of Chemistry, Faculty of Science, Sts. Cyril and Methodius University, 
P.O. Box 162, 1001 Skopje, Republic of Macedonia;
A high-performance liquid chromatographic (HPLC) method for determination of ofloxacin and lomefloxacin
in human plasma has been developed and validated. The developed HPLC method employing protein precipitation
for sample preparation is simple and convenient for the simultaneous determination of ofloxacin and lomefloxacin
in plasma samples. For the purpose of minimizing the variability caused by sample pretreatment a method of inter-
nal standardization for the quantification of these drugs is suggested. 
In order to fulfil the aim, the method was first developed for the separation of and determination of quinolones
concentrations by optimizing the experimental parameters and determining linearity for the two fluoroquinolones. Effect
of organic modifier on the retention of investigated drugs was investigated. A simple isocratic chromatographic assay
with UV-detection at 280 nm was performed on Hibar Lichrospher 100 RP 8 coloumn (250 x 4.6 mm, 5 
µm, Merck,
Germany) using acetonitrile and 0.5 % triethyl amin in water (pH to 2.5 adjusted with H
3
PO
4
) mixture (15:85, V/V) as
a mobile phase at a flow rate of 1.2 mL min-
1
. It was found that there is no need to develop a method which can pro-
vide the selective determination of ofloxacin and its microbiologically active metabolite (desmethyl ofloxacin)
because ofloxacin is not extensively metabolized and only low concentrations of active ofloxacin metabolite are
attained in serum. 
The method for the determination of ofloxacin and lomefloxacin concentrations was validated by evaluating
recovery, selectivity, linearity, precision and accuracy. The calibration curves were linear in the concentration range of
0.5-6.0 µg mL-
1
for ofloxacin and 0.2-4.5 µg mL-
1
for lomefloxacin. The limit of quantification defined as the lowest
amount detectable with a precision of less than 15 % and an accuracy of ±15 % was found to be 0.5 µg mL-
1
for
ofloxacin and 0.2 µg mL-
1
for lomefloxacin. It was shown that the method can be successfully used to monitor ofloxacin
and lomefloxacin levels in clinical samples.
Macedonian pharmaceutical bulletin 53 (1,2) 183-184 (2007)
PP - 82
183
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Razvoj i validacija na HPLC metoda za opredeluvawe 
na Ofloksacin i Lomefloksacin vo humana plazma
Dragica Zendelovska
1
, Traj~e Stafilov
2
1
Institut za predklini~ka i klini~ka farmakologija i toksikologija, Medicinski fakultet,
Univerzitet “Sv. Kiril i Metodij”, 50 Divizija bb, 1000 Skopje, Makedonija; 
2
Institut za hemija, Prirodno-matemati~ki fakultet, 
Univerzitet “Sv. Kiril i Metodij”, p. fah 162, 1001 Skopje, Makedonija;
Razrabotena e i validirana metoda za opredeluvawe na ofloksacin i lomefloksacin vo humana
plazma so primena na visokoefikasna te~na hromatografija (HPLC). Metodata e ednostavna i pogodna za
simultano opredeluvawe na ofloksacin i lomefloksacin vo plazma. Pri podgotovkata na primerocite
predvidena e precipitacija na proteinite. So cel da se minimiziraat razlikite pri podgotovkata na pri-
merocite predlo`en e metod na vnatre{na standardizacija pri kvanifikacijata na ovie lekovi. 
Pri razrabotkata na postapkata za separirawe i opredeluvawe na koncentraciite na hinolonite
fluorohinoloni. Ispituvano e vlijanieto na ogranskite modifikatori na retencionoto vreme na ispituva-
nite lekovi. Hromatografskoto razdeluvawe na ofloksacin i lomefloksacin (pri UV detekcija na 280 nm)
e izvedeno na Hibar Lichrospher 100 RP 8 kolona (250 x 4,6 mm, 5 
µm, Merck, Germany) so primena na smesa od
acetonitril i 0,5 % trietilamin vo voda so pH=2,5 (15:85, V/V) kako mobilna faza pri protok od 1,2 mL min-
1
.
Utvrdeno e deka ne e potrebno da se razrabotuva posebna metoda koja }e ovozmo`i selektivno opredeluvawe na
ofloksacinot i negoviot mikrobiolo{ki aktiven metabolit (desmetil ofloksacin) zatoa {to ofloksaci-
not ne se metabolira intenzivno i aktivniot metabolit e prisuten vo serumot vo mnogu niski koncentracii.
Metodata za opredeluvawe na ofloksacin i lomefloksacin e validirana preku evaluacija na anal-
iti~kiot prinos, selektivnosta, lineranosta, preciznosta i to~nosta. Kalibracionite dijagrami se lin-
earni vo koncentraciono podra~je od 0,5 do 6,0 µg mL-
1
za ofloksacin i od 0,2 do 4,5 µg mL-
1
za lomeflok-
sacin. Granicata na kvantifikacija, definirana kako najmalo koli~estvo {to mo`e da se detektira so
preciznost pomala od 15 % i to~nost od 15 %, iznesuva 0,5 µg mL-
1
za ofloksacin i 0,2 µg mL-
1
za lome-
floksacin. Poka`ano e deka metodata mo`e da se primenuva za sledewe na ofloksacin i lomefloksacin
vo klini~ki primeroci. 
Macedonian pharmaceutical bulletin 53 (1,2) 183-184 (2007)
PP - 82
184
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Development of a chromatographic bioanalytical method for the assy 
of Cefotaxime and its metabolyte in human urine
M. Ze
c
evi
c
1
, M. M. Aleksi
c
2
, V. Kapetanovi
c
3
, B. Joci
c
1
, J. Atanackovi
c
3
Institute of 
1
Pharmaceutical, 
2
Physical and 
3
Analytical Chemistry, Faculty of Pharmacy,
University of Belgrade, Belgrade, Serbia
Cefotaxime sodium is a semisynthetic third generation cephalosporin antibiotic used for the treatment of
infections caused by a wide variety of Gram-negative and Gram-positive species of bacteria. In the human organ-
ism, it is metabolized by esterases to desacetylcefotaxime, active metabolite, and several non-active metabolites.
Various methods and reviews have been published covering the analysis of cephalosporins in biological
matrices. Only few of the reported methods in literature determine simultaneously cefotaxime and desacetylcefo-
taxime in human plasma and cerebrospinal fluid by high-performance liquid chromatography and no literature data
was found considering their simultaneous determination in human urine [1-4]. Chromatographic analysis of men-
tioned substances appeared to be quite difficult, due to their very polar nature and instability.
This study presents a simple, rapid, accurate and sensitive RP HPLC method developed and validated for
the assay of cefotaxime and desacetylcefotaxime in human urine. The assay involved deproteinisation of the urine
sample and subsequent separation of analytes on a C
18
reversed-phase HPLC column, with ultraviolet detection at
262 nm. Satisfactory separations were achieved with the mobile phase consisting of the mixture of acetonitrile and
0.007M orthophosphoric acid (15:85 v/v) pumped at a flow rate of 1 ml/min. Cefotaxime and desacetylcefotaxime
eluted with retention times of 4.057 and 1.960 min, respectively. The proposed composition of the mobile phase was
suitable to allow the appropriate retention of investigated substances on the column in order to avoid all the inter-
fering peaks or the co-elution of the complex urine matrix.
The proposed chromatographic method was validated in accordance with FDA bioanalytical method vali-
µg/ml for cefotaxime and 1.10–11.00 µg/ml for desacetylcefotaxime). These ranges were selected in accordance
with the pharmacokinetic profile of cefotaxime and could be appropriate even when dealing with the patients with
impaired renal function. Following the method validation procedure, all the obtained results were in accordance with
the demanded acceptance criteria proving in that manner that the proposed bioanalytical method could be used in a
routine laboratory practice.
[1] K.B. Patel, D.P. Nicolau, C.H. Nightingale, R. Quintiliani, Pharmacol. 22 (1995) 49.
[2] T. Scanes, A.F. Hundt, K.J. Swart, H.K.L. Hundt, J. Chromatogr. B 750 (2001) 171.
[3] E. Yun, A. Prince, J. McMillin, L. Welch, J. Chromatogr. B 712 (1998) 145.
[4] A. El-Gindy, A. El Walily, M. Bedair, J. Pharm. Biomed. Anal. 23 (2000) 341.
[5] Guidance for industry: Boianalytical method validation, US department of Health and Human service, 
Food and Drug Administration, CDER, Rockville, MD, USA, 2001.
Macedonian pharmaceutical bulletin 53 (1,2) 185 (2007)
PP - 83
185
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Determination of Thioctic (
α-lipoic) acid by derivate 
UV - spectrophotometry
Zagorka Koricanac, Tatijana Jovanovic, Sladjana Tanaskovic
Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia,,
Thioctic acid (
α-Lipoic acid), 1,2-dithiolane-3-pentanoic acid, is used extensively in the treatment of vari-
ous diseases, such as alcoholic liver disease, mushroom poisoning, heavy metal poisoning, glaucoma, radiation injury
and neurodegenerative disorders; it has been proposed that thioctic acid acts as an antioxidant and interferes with
the pathogenesis of diabetic polyneuropathy. The kinetic spectrophotometric method was applied for determination
of thioctic acid in pharmaceutical dosage forms (tablets) and human serum, based on the catalytic effect of this com-
pound on the iodine-azide reaction. In earlier work we developed spectrophotometric methods for determination of
thioctic acid in water and pharmaceutical dosage forms with palladium(II) chloride as reagent. Continuous our stud-
ies, in the present work suitable conditions for direct second derivative spectrophotometry determination of thioctic
acid in water solution were established; sensitive and reproducible spectrophotometric method for the determination
of thioctic acid in injection solution (Berlithion
®
300 ED ampoules containing 300 mg thioctic acid/12 ml (Berlin-
Chemie Germany), was reported. Absorption spectra and spectrophotometer measurements were carried out on a
GBC, UV/VIS, Contra 20 double-beam spectrophotometer (Australia), in 1cm quartz cuvettes (a slit width of 1 nm,
scan speed 200 nm/min, time response 0,1 ms, 
∆λ=2 nm). For pH measurements, Radiometer PHM 62 pH meter
(Copenhagen), calibrated with appropriate standard buffer solutions, was utilized for pH measurements.
UV absorption spectra of thioctic acid were determined of various pH values. The shape of absorption spec-
trum and position of absorption maximum dependence of pH (2-10); in acid medium exists one maximum of
absorbance, and the other in alkaline medium. Both of them can be used for analitical determination. The second
order derivative UV-spectrum was chosen for analysis because the absorbance band in the zero order spectrum of
investigated thioctic acid is broad, in acid medium. The calibration graph was constructed by plotting concentration
versus peak-to-peak amplitude in the second-derivative UV spectrum between 200-350 nm. All parameters for valida-
tion of the proposed method are given.
The proposed method is simple and fast, allow precise and accurate results, and it can be applied to the assay
of small amounts of investigated thioctic acid.
Macedonian pharmaceutical bulletin 53 (1,2) 186 (2007)
PP - 84
186
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Determination of Salbutamol sulphate residues on manufacturing 
equipment surfaces in cleaning validation proces
Samira Omerovic, A. Dizdarevic, L. Mujkic, M. Buturovic
Quality Control Department, Bosnalijek, Pharmaceutical and Chemical Industry, Joint Stock Company, 
71000 Sarajevo, 53 Jukiceva Str., Bosnia and Herzegovina
cGMP, FDA and ICH regulations have resulted in cleaning validation becoming an essential part of the pro-
duction of drugs and active pharmaceutical ingredients.
In many cases, the same equipment may be used for processing different products. 
To avoid contamination of the following product, adequate cleaning procedures are essential. Pharmaceutical
products and active pharmaceutical ingredients (APIs) can be contaminated by other pharmaceutical products, by
cleaning agents, micro-organisms or by other material. Further sources of contamination might be raw materials,
intermediates, products of degradation, etc.
Validation of cleaning procedures is thus critical to any Quality Assurance programme and is currently topis
of great concern to regulators worldwide. 
According to definition:“ Cleaning Validation is a process to ensure that equipment cleaning procedures are
removing residues to predetermined levels of acceptability.” 
In this research a sensitive analytical method based on high performance liquid chromatography, was adapt-
ed and validated to determine residues of salbutamol sulphate during the process of cleaning validation.
The detection limit for proposed liquid chromatography analytical method was  sufficiently sensitive to detect
the established acceptable level of the residues of salbutamole. Acceptance critera was from 0.65 ppm to 3 ppm per ml.
In the paper, direct sampling method from equipment contact surface, was also presented.
As sensitive sampling methods require development and must be applicable to each specific piece of equip-
ment used, swab recovery was determined using spiking studies incorporating coupons of equipment surfaces. The
swabbing procedure was optimized in order to obtain a suitable recovery of salbutamol from Stainless Steel, Aluminium
and Plexiglass
A mean recovery factor close to 90%, show that applied sampling method is acceptable for cleaning valida-
tion study.
Macedonian pharmaceutical bulletin 53 (1,2) 187 (2007)
PP - 85
187
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Development and validation of the HPLC method 
for related substances in Doxazosin tablets
A. Nalo, E. Dizdarevic, E. Vranjes, S. Hadzidedic
Bosnalijek d.d., Development Department, Jukiceva 53, 71000 Sarajevo, Bosnia and Herzegovina
A simple and reversed-phase high performance liquid chromatography method for separation and determi-
nation of related supstances of Doxazosine Mesylate in tablets has been developed. The separation of related sup-
stances of Doxazosin has been achieved on a Lichrosper 100 RP 18 column (4,6x250 mm;5 µm) as stationary phase,
and using buffer pH 5.0, acetonitrile and methanol (400:300:300) as mobile phase at a flow rate of 1.0 ml/min.
Detection was carried out using a photo diode array detector at 225 nm.
To declare the method as stability indicating, degradation of active compound was performed. Degradation
conditions included acid hydrolysis, alkaline hydrolysis, oxidation, thermal degradation of solid active substance, ther-
mal degradation of active substance in solution and degradation of active compound under the influence of daylight.
The method was validated with respect to accuracy, precision,linearity and its limits of detection and quan-
tification.
The chromatographic behavior of all the compounds was examined under variable compositions of differ-
ent ratio of solvents in mobile phase, temperatures, buffer concentrations and pH values, wavelength, column and
flow rate.The parameter measured : retention time, selectivity and resolution. 
The method could detect the related supstances at level of 0,025 
µg/ml for related substance 1 and 0,051
µg/ml for related substance 2 and could quantified the related supstances at level 0,075 µg/ml for related substance
1 and 0,15 
µg/ml for related substance 2.
Coefficient of variation for precision are 0,40 for Doxazosin related substance 1 and 1,39 for Doxazosin
related substance 2.
Coefficient of variation for accuracy for Doxazosin related substance 1 is 1,31 and for Doxazosin related
substance 2 is 3.18.
The correlation coefficient of linearity for other related substances was 0.99898, for related substance 1 it
was 0,99999 and for related substances 2 it was 0,99989.
The standard curves were linear over the concentration ranges, 0.2 to 4 µg/ml for Doxazosine Mesylate,
Doxazosine related substance 1 and related substance 2.
This study shows that the proposed method is accurate, linear, precise and sensitive for the determination of
Doxazosine Mesylate related substances.
Macedonian pharmaceutical bulletin 53 (1,2) 188 (2007)
PP - 86
188
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The comparison of dissolution profile for Lisinopril 
and hydrochlorothiazide tablets
N. Avdic, G. Dragosevic, E. Vranjes, S. Hadzidedic
Bosnalijek d.d., Development Department, Jukiceva 53, 71000 Sarajevo, Bosnia and Herzegovina
A combination of angiotensin-converting anzyme (ACE) inhibitor, Lisinopril and diuretic, Hydrochloro-
thiazide, enables additive antihypertensive action. As a result of its diuretic effects, Hydrochlorothiazide increases
plasma rennin activity, increases aldosterone secretion, and decreases serum potassium. Administration of Lisinopril
blocks the rennin-angiotensin aldosterone axis and tends to reverse the potassium loss associated with the diuretic.
Lisinopril is chemically described as (S)-1-[N
2
-(1-carboxy-3-phenylpropyl)-L-lysyl]-L-proline dihydrate,
and Hydrochlorothiazide is 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadi-azine-7-sulfonamide 1,1-dioxide.
Since the manufacturers of Lisinopril Dihydrate and Hydrochlorothiazide were changed, it was mandatory
to perform testing to prove that this change does not have an impact on our product. One of the ways to prove that
is to test dissolution profiles of tablets that have incorporated APIs made by new manufacturers and to compare it
with the dissolution profiles of the tablets with the API produced by the old manufacturer.
In vitro dissolution (content release) of Lisinopril and Hydrochlorothiazide was performed according to gen-
eral procedure USP <711> apparatus 2, Method of rotating paddle. Use 0.1 mol/l of hydrochloric acid, 900 ml as a
medium, at temperature of 37 
0
C 
± 0.5 
0
C, with mixing speed 50 rpm. Sample medium was taken every 5 minutes
with complete test duration of 30 minutes, and Lisinopril and Hydrochlorothiazide assay is to be determined by
HPLC method.
As stationary phase LiChrospher
®
100 RP- 8 column; length of 250 mm, internal diameter 4 mm and 5 
µm
particle size was used. A mixture of 850 ml of phosphate buffer pH 2.4-2.5 and 150 ml of Acetonitrile was used as
mobile phase, with a flow rate of 1.5 ml / min. Column temperature was maintained at 50oC and injection volume
of sample was 20 
µl. Detection was performed at 210 nm.
For curves to be considered similar, f
1
values should be close to 0, and f
2
values should be close to 100.
Generally, f
1
values up to 15 (0-15) and f
2
values greater than 50 (50 -100) ensure sameness or equivalence of the
two curves and this of performance of the test (post change) and reference (prechange) products.
In our cases, factors of similarity f
2
are greater then 60%, and factors of difference f
1
are less then 10%, so
it can be concluded that change of manufacturer active components does not have impact on dissolution profile. 
Macedonian pharmaceutical bulletin 53 (1,2) 189 (2007)
PP - 87
189
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Determination of Clindamicin in phermaceutical formulation 
by capillary electrophoresis
Z. Kavrakovski, Z. Kitanovski, K. Mladenovska
Faculty of Pharmacy, University „Ss. Cyril and Methodius“, Vodnjanska 17, Skopje, Macedonia
Capillary zone electrophoresis (CZE) is generally considered as a complementary or alternative technique
to HPLC for pharmaceutical analysis. The popularity of CE in the pharmaceutical field has been accelerated by its
simplicity, high efficiency and selectivity, large separation capacity, including faster analysis and method develop-
ment, lower consumable expenses and easier operation. CE has been applied to all major drug classes and specific
reviews have been published on the application of CE to the analysis of antibiotics. Clindamycin (including the HCl
salt and other forms) is a common antibiotic that is marketed for the treatment of certain Gram-positive bacterial
infections in a form of capsules, topical solutions, lotions, gels and creams. Several methods have been published
for the determination clindamycin in bulk drugs and formulations, including microbial and spectrophotometric assays,
GLC, HPLC. Refractive index, electrochemical and UV detection have been also applied. Some of the methods suf-
fer from luck of specificity and accuracy, others require complicated sample manipulation. UV detection seems to
offer more, in sense of sensitivity and stability. This work describes the development and validation of a sensitive
and relatively rapid CZE method with UV detection for determination of clindamycin after dissolving in aqueous
solutions with various pHs ranging from 2-5, where clindamycin showed maximum stability. From previous expe-
rience it was known that pKa of clindamycin is about 7.6. For this reason, a running electrolyte solution pH well
below the pKa was chosen.
For the experiments BioRad system model (BioFocus 3000, USA, on-column diode array UV absorbance
detector at 190 nm; BioRad software Ver.5.2 for Windows NT) was used. Untreated fused silica capillaries (BioRad,
USA), inner and outer diameter 100 and 375 
µm, respectively, and a total length of 30 cm (25 cm to the detector)
were used. Aqueous solutions of clindamycin were introduced by hydrodynamic injection for 5 sec at the anodic end
of the capillary at a constant temperature of 30
0
C. Running electrolyte solution was 0.07 M phosphate, pH 3.28
adjusted by 0.1 M phosphoric acid. Separations were performed at 11.0 kV. The optimum capillary rinse procedure
was 1 min rinse with phosphoric acid 0.05 M and 1 min rinse with the running buffer. Under these conditions, base-
line separation of clindamycin was achieved in less than 10 min with migration time RSDs from 0.18 to 0.30% (n=6).
The standard curves were linear over the concentration range of 0.007-1.01 mg/ml. The limit of quantification was
0.14% (m/m). The method showed good validation data in terms of precision, limits of quantification and detection,
specificity and linearity and was found to be suitable for analysis of clindamycin hydrochloride in solutions with a
recovery of 95.6%-98.9% when clindamycin capsules were analyzed. The proposed CE method with UV detection
is a simple, fast and robust method for the analysis of clindamycin in its pharmaceutical dosage forms that involves
very little sample preparation. Quantitative analysis indicates potential usefulness of capillary electrophoresis as an
alternative to the assay method prescribed in the Ph Eur and USP.
Macedonian pharmaceutical bulletin 53 (1,2) 190-191 (2007)
PP - 88
190
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