ISSN 10619348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 6, pp. 545–551. © Pleiades Publishing, Ltd., 2013.

ARTICLES

Development and Validation of an HPLC Method

for the Determination of Verapamil Residues in Supports
of Cleaning Procedure1 ˆ ˆ
Dragan M. Milenovica, Sne z ana P. Milo s evica, Svetlana Lj. Durica,
ˆ ˆ
Daniela C . Naskovica, and Sne z ana S. Miticb
a
“ZdravljeActavis” company, R&D Vlajkova street 199, Leskovac, 16000 Serbia ˆ
b Faculty of Sciences and Mathematics, Department of Chemistry, University of Ni s
ˆ ˆ
Vi s egradska 33, P.O. Box 224, Ni s , 18000 Serbia
Received March 23, 2011; in final form, June 17, 2011

Abstract—Analytical method validation, determining the recovery rate from the equipment surface, and sta
bility of a potential contaminant are important steps of a cleaning validation process. An HPLC method for
the determination of the verapamil residues on stainless steel surfaces of the equipment employed in drug
manufacture is described. The cleaning validation sample impurities as well as excipients of the commercial
sample did not interfere in the analysis which proved the selectivity of the method. The validation of the method
demonstrated acceptable levels of the linearity, precision and accuracy. Cotton swabs, moistened with methanol
were used to remove any residues of drugs from stainless steel surfaces, and give recoveries of above 78.59% for three
diferent concentration levels. The precision of the results, reported as the relative standard deviation (RSD, %),
were below 1.58%. Low quantities of the drug residues were determined by HPLC using a Hypersil ODS column
(125 × 4.0 mm, 5 µm) at 25°C with the mobile phase metanol–water⎯triethylamine (70 : 30 : 0.2, v/v/v) at a flow
rate of 0.6 mL/min, injection volume of 50 µL and detection at 278 nm.

Keywords:cleaning validation, verapamil, swab analysis, residues

DOI: 10.1134/S1061934813060051

1 Pharmaceutical manufacturing equipment has to
be cleaned after production in order to avoid cross
contamination in the next batch of a different product.
In the end of the cleaning procedure the effectiveness
of the cleaning is checked using a validated analytical
method suitable to investigate the traces of residues.
The cleaning validation consists of two separate
steps: the first one is the development and validation of
the cleaning procedure, which is used to remove drug
residue from the manufacturing surfaces, and the sec
ond one involves the developing and validating of the
methods for quantifing residuals from surfaces of the
manufacturing equipment. It is the responsibility of
the manufacturer to develop robust cleaning proce
dures, and to demonstrate that execution of the clean
ing procedures was successful. Futhermore, many
sampling points of the manufacturing equipment have
to be tested for verifying occurrence of contamination.
For these reasons, an analytical method for residue
monitoring should also be rapid and simple [1].
The acceptable limit for residue in the equipments
is not established in the current regulations. The
design of a suitable sampling procedure and analytical
method is very important in cleaning validation. The
1 The article is published in the original.
technique must be appropriate for measuring the ana
lytes at and below the residue acceptable limit.
According to FDA (Food and Drug Administration),
the limit should be based on logical criteria, involving
the risk associated with residues of a determined prod
uct. The calculation of acceptable residual limit, max
imum allowable carryover, for active products in pro
duction equipments should be based on therapeutical
doses, toxicological index and a general limit (10 ppm)
[1–4].
Verapamil, [(±)5[N(3,4dimethoxyphenethyl)
Nmethylamino]2(3,4dimethoxyphenyl)2isopro
pylvaleronitrile], is a calciumchannel blocker and is
classified as a class IV antiarrhythmic agent. It is used in
the control of supra ventricular tachyarrhythmias, and
in the management of classical and variant angina pecto
ris [5].
Numerous methods have been reported for the
quantitative determination of verapamil hydrochlo
ride in the raw materials [6–13], tablets and other
solid dosage forms [5, 14–16], human plasma [17], by
HPTLC or TLC [18, 19]. A literature review revealed
that no validation of cleaning methods for verapamil
could be found.

545
546
DRAGAN M. MILENOVIC
Taking the above mentioned consideration into ac
count, the aim of this study was to develop and validate
simple analytical method that allows the determina
tion at trace level of residual verapamil in production
area equipment and to confirm efficiency of cleaning
procedure. The analytical method proposed has been
validated considering selectivity, linearity, accuracy,
precision and limits of detection (LOD) and quantita
tion (LOQ). The stability of verapamil samples was al
so studied [20].
EXPERIMENTAL
Chemicals and reagents. The verapamil hydrochlo
ride, working certified standard, was purchased from
Recordati, Industria Chimica E Farmaceutica S.p.A.
(Italy). Methanol (HPLC gradient grade) and triethyl
amine were purchased from J.T. Baker (Deventer,
Holland). Purified water was obtained with a Arium
Laboratory Equipment (RO, UV) by Sartorius AG
(Gottingen, Germany). The extractionrecovery sam
pling was done with Alpha® Swab polyester on
polypropylene handle—TX714A (ITW Texwipe®,
Mahwah, NJ, USA). The mobile phase was filtered
through a 0.45 µm Sartorius membrane filter (Gottin
gen, Germany).
Equipment. The HPLC system consisted of a de
gasser G1379B, a bin pump G1312A, an automatic in
jector G1329A, a thermostated column compartment
G1316A and a multiwavelength detector G1365B
(multiwavelength), all 1200 Series, from Agilent Tech
nologies controlled by an HP Chemstation software
(Waldbroon, Germany). Ultrasonic bath was from Elma,
Transsonic 470/H (Singen, Germany). Analytical bal
ance was from Sartorius AG, CP224SOCE (Gottingen,
Germany); accuracy of the balance: ±0.0001 g.
Chromatographic conditions. All chromatographic
experiments were performed in the isocratic mode.
The mobile phase was constituted of methanol–wa
ter–triethylamine (70 : 30 : 0.2, v/v/v), at a flow rate
of 0.6 mL/min. UV detection was made at 278 nm.
The volume of injection was fixed at 50 µL. All analy
ses were performed at 25°C. The separation was car
ried out on a Hypersil ODS column (125 × 4.0 mm,
5 µm) from Agilent.
Standard solutions preparation. Stock solution of
standard was prepared by accurately weighing vera
pamil hydrochloride standard (25 mg ± 0.1 mg), trans
ferring it into 25 mL volumetric flask, diluting to vol
ume with methanol, and sonicating for 15 min. Dilu
tions were later prepared with the mobile phase to obtain
the solutions for calibration (2.50 do 50 µg/mL) and
standard solution for the positive swab control at three
concentration levels (50, 100, and 150 µg/swab level).
These solutions were filtered through a 0.45 μm regen
erated cellulose filter and injected in triplicate.
Sample preparation. The selected surfaces
(5 × 5 cm) of stainless steel, previously cleaned and
JOURNAL OF ANALYTICAL CHEMISTRY
' et al.
dryed, were sprayed with 500 µL of a standard solu
tions for positive swab control at all concentration lev
els, and the solvent was allowed to evaporate (approx
imate time was 2 h). The surfaces were wiped with the
first cotton swab soaked with methanol, passing it in
various ways, to remove the residues from stainless
steel. The other dry cotton swab was used to wipe the
wet surfaces. The swabs were placed into the 25 mL
screw cap test tubes, and 5.0 mL of the mobile phase
was pipetted into adequate sample tubes. The back
ground control sample was prepared from the extrac
tion media. The negative swab control was prepared in
the same way as the samples, using swabs, which had
not been in contact with the test surface. Also, the test
and excipient solutions were prepared according to the
content of tablets to assure that they did not interfere
with verapamil hydrochloride. After that, the tubes
were placed in the ultrasonic bath for 30 min and the
solutions were analysed by HPLC. FDA guidelines
recommend a minimum recovery of 50%.
RESULTS AND DISCUSSION
Acceptance limit calculation. The maximum allow
able carryover—MACO is acceptable transferred
amount from the previous to the following product.
MACO is determined on the basis of therapeutic dose,
toxicity and general 10 ppm criterion. The next step is
to determine the residue limit per surface area from
the equipment surface area and the most stringent
maximum allowable carryover (the most stringent cri
terion being based on the therapeutical dose in this case).
The calculated limit per surface area in the case of vera
pamil hydrochloride was 100 µg/swab for 25 cm2.
Selection of the chromatographic conditions. To ob
tain the best chromatographic conditions, the wave
length for detection, the column and the mobile phase
composition were adequately selected. The main ob
jective was to develop a liquid chromatographic meth
od that, working in isocratic mode, allowed us to de
termine the total verapamil hydrochloride residues
collected by the swabs, without interference of impu
rities which originated from swabs, plates, extraction
media.
The wavelength of 278 nm was selected for the
analysis because the drug had sufficient absorption
and low quantities of verapamil hydrochloride may be
detected correctly. Furthermore, the calibration
curves obtained at 278 nm show good linearity.
Starting point for the development of the cleaning
assay for verapamil hydrochloride was modified work
on the assay method for verapamil in capsules [14] by
using Purospher STAR RP18e, 250 × 4 mm, 5 µm
column with mobile phase acetonitrile–methanol–
phosphate buffer (the buffer was prepared with 0.025 M
potassium dihydrogen phosphate by adjusting to pH 3.0
with ophosphoric acid) (40 : 40 : 20 = v/v/v), with
50 µL injection volume at 278 nm. An initial attempt
Vol. 68 No. 6 2013