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Development and validation of an HPLC method for the determination of verapamil residues in supports of cleaning procedure

Authors:

Abstract

Analytical method validation, determining the recovery rate from the equipment surface, and stability 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.
ISSN 10619348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 6, pp. 545–551. © Pleiades Publishing, Ltd., 2013.
545
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(3,4dimethoxyphenethyl)
N
methylamino]2(3,4dimethoxyphenyl)2isopro
pylvaleronitrile], is a calciumchannel blocker and is
classified as a class IV antiarrhythmic 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.
Development and Validation of an HPLC Method
for the Determination of Verapamil Residues in Supports
of Cleaning Procedure
1
Dragan M. Milenovic
a
, Sne ana P. Milo evic
a
, Svetlana Lj. uric
a
,
Daniela . Naskovic
a
, and Sne ana S. Mitic
b
a
“ZdravljeActavis” company, R&D Vlajkova street 199, Leskovac, 16000 Serbia
b
Faculty of Sciences and Mathematics, Department of Chemistry, University of Ni
Vi egradska 33, P.O. Box 224, Ni , 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
µ
m) 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
µ
L and detection at 278 nm.
Keywords
:cleaning validation, verapamil, swab analysis, residues
DOI:
10.1134/S1061934813060051
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ARTICLES
546
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 6 2013
DRAGAN M. MILENOVIC
'
et al.
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 extractionrecovery 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
µ
m 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, CP224SOCE (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
µ
L. All analy
ses were performed at 25
°
C. The separation was car
ried out on a Hypersil ODS column (125
×
4.0 mm,
5
µ
m) 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
µ
g/mL) and
standard solution for the positive swab control at three
concentration levels (50, 100, and 150
µ
g/swab level).
These solutions were filtered through a 0.45
μ
m regen
erated cellulose filter and injected in triplicate.
Sample preparation.
The selected surfaces
(5
×
5 cm) of stainless steel, previously cleaned and
dryed, were sprayed with 500
µ
L 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
µ
g/swab for 25 cm
2
.
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 RP18e, 250
×
4 mm, 5
µ
m
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
o
phosphoric acid) (40 : 40 : 20 = v/v/v), with
50
µ
L injection volume at 278 nm. An initial attempt
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 6 2013
DEVELOPMENT AND VALIDATION OF AN HPLC METHOD 547
resulted with short retention time (about 4 min), USP
tailing about 1.7, but recovery value for the lowest con
centration level were above 102% (102–106%) (inter
ferences with swab samples).
Therefore, our work were proceeded with Hypersil
ODS short column (125
×
4 mm, 5
µ
m) in order to get
the shortest retention time, with mobile phase con
taining a considerable amount of organic modifier,
methanol
water
triethylamine (70 : 30 : 0.2, v/v/v).
The peak symmetry was improved by the addition of a
triethylamine into the mobile phase. Retention time
was about 9 min, with excelent features of peaks (USP
tailing about 1.0). Concerning not too long retention
time and excelent features of peaks, this chromato
graphic conditions were used for the rest of the work.
Triethylamine acts as a competing base and minimizes
solutesilanol interactions.
The injection volume was set at 50
µ
L in order to
increase the responce of the method without sacrific
ing chromatographic peak shape. Also, flow rate was
set at 0.6 mL/min in order to get higher responce of
the metod (lower flow—lower column backpressure,
higher peak area). As increase in temperature did not
affect on chromatographic efficiency (number of the
oretical plates), a temperature of 25
°
C was selected.
Taking into account the results obtained with dif
ferent columns and mobile phases assayed, finally we
have chosen chromatographic conditions which were
mentioned because the quantification limits obtained
were the lowest, with good sensitivity and without
interferences.
Sample treatment optimization.
Cotton swabs were
spiked with 150
µ
g/swab of verapamil hydrochloride
and were placed into a tube. After adding the different
solvents (water, methanol and the mobile phase), the
tube was sonicated in different times (5, 15 and 30 min)
and the solutions were analyzed by HPLC. The opti
mum condition was achieved with the mobile phase as
an extracting solvent and the best sonication time was
30 min. Results are given in Table 1.
In all cases, the best results were obtained using the
two cotton swabs for sampling (the first one was wetted
with methanol and the second one was dry), so this
technique was used for the rest of the work. Results are
given in Table 2.
Surface for swabbing.
Stainless steel coupon was an
obvious choice for surface material, because more
than 95% of manufacturing equipment surfaces are
stainless steel. For practical reasons, coupon dimen
sions of 5
×
5 cm were chosen. Recovery % for differ
Tabl e 1 .
Results of sample treatment optimization (recovery % ± confidence interval;
n
= 3)
Analite Solvent Volume, mL
Time of extraction, min
51530
Verapamil hydrochloride Mobile phase 5 91.23 ± 2.61 93.34 ± 1.56 96.39 ± 0.18
10 92.71 ± 1.15 93.21 ± 2.43 97.21 ± 1.32
Methanol 5 84.41 ± 1.54 85.32 ± 2.36 85.52 ± 2.11
10 85.24 ± 2.34 86.21 ± 3.31 87.51 ± 0.87
Water 5 82.65 ± 2.12 83.74 ± 1.15 83.91 ± 2.75
10 83.24 ± 3.54 84.51 ± 2.71 84.62 ± 0.18
Table 2.
Recovery (%) for different swab numbers used
Analite Concentration,
µ
g/swab (
n
= 3)
Sampling method
Swab wetted by methanol Swab wetted by methanol,
afterwards 1 dry swab
Verapamil hydrochloride 50 75.73 ± 2.14 93.04 ± 1.48
100 78.56 ± 2.31 95.40 ± 0.05
150 80.11 ± 4.31 96.39 ± 0.18
548
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 6 2013
DRAGAN M. MILENOVIC
'
et al.
ent surface areas (glass and polivinylplastics) are also
investigated, regardless of their little part in total area.
Results are given in Table 3.
Validation of the chromatographic method
. Once
the chromatographic conditions had been selected,
the method was validated paying attention to selectiv
ity, linearity, limit of detection, limit of quantification,
precision, accuracy and sample stability.
Suitability test.
System suitability testing is essen
tial for the assurance of the quality performance of the
chromatographic system. During performing system
suitability tests, in all cases, % RSD for peak areas was
<2%, the average number of theoretical plates per col
umn was >3300, the USP tailing factor was about 1.0.
Selectivity
. Selectivity has been checked by inject
ing a standard of verapamil hydrochloride, the back
ground control sample containing the mobile phase,
the negative swab control, the unspiked stainless steel
5
×
5 cm plate swabbed as described, the excipient
mixture. In figure, it can be observed that there are no
mutual interferences.
Linearity
. Linearity of the method was studied by
analyzing the standard solutions at different concen
tration levels ranging from 2.50 to 50.00
µ
g/mL, with
triplicate determination at each level. The calibration
curve was constructed by plotting the mean response
area against the corresponding concentration inject
ed, using the least square method. Values of the slope,
the intercept and coefficient of determination of the
calibration curve for verapamil hydrochloride are giv
Table 3.
Recovery (%) for different surfaces for lowest concentration level
Analyte Surface Concentration,
µ
g/swab Recovery % ± CI*
Verapamil hydrochloride Stainless steel 93.04 ± 1.48
Glass 50 86.14 ± 1.56
Polivinylplastics 84.21 ± 3.41
*Confidence interval.
(a)
(b)
10
30
25
20
15
5
01486104
212
Time/min
Abs/mAU
Verapamil
8.639
10
30
25
20
15
5
01486104
212
Abs/mAU
10
30
25
20
15
5
01486104
212
Abs/mAU
(с)
Chromatograms obtained for: (a) non–spiked stainless steel surface, (b) excipient mixture, (c) standard solution of verapamil
hydrochloride (20
µ
g/mL).
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 6 2013
DEVELOPMENT AND VALIDATION OF AN HPLC METHOD 549
en in Table 4. The high value of the coefficient of de
termination indicated a good linearity.
Limits of detection and quantitation
. LOD and
LOQ were determined based on the standard deviation
of the response (
y
intercept) and the slope of the cali
bration curve according to the ICH guidelines. LOD
and LOQ for verapamil hydrochloride were found to
be 1.64 and 4.98
µ
g/mL, respectively (Table 4).
Precision and accuracy.
The precision and accura
cy of the chromatographic method, reported as rela
tive standard deviation (RSD %) and the recovery %,
respectively were assessed by estimating the repeat
ability of the results for six replicate injections at three
different concentration levels. The method precision
and accuracy was determined on the spiked and dried
swabs and plates. The recovery, 95% confidence inter
val, and RSD values obtained on the spiked and dried
swabs and plates (Table 5) per each level illustrated
good precision and accuracy of the method. These
precision and recovery results are acceptable for the
purpose of residue monitoring.
Intermediate precision of the method was investi
gated by making five consecutive injections of a stan
dard solutions in two different days with different ana
lysts, on two different HPLC systems. On both days
the RSD % were calculated for peak area responses
obtained for the verapamil hydrochloride peaks. The
data obtained suggested that the method exhibited an
acceptable intermediate precision with less than 2.0%
RSD for the verapamil hydrochloride standard solu
tion.
Sample stability.
The stability of the verapamil hy
drochloride in the swab matrix was tested. The spiked
samples at all concentration levels were stored after
analyses in the injector vials in the autosampler tray at
ambient temperature for 7 days. All the samples were
injected into the appropriate HPLC system after 24 h,
48 h and 7 days against fresh standard solutions. No
changes in the chromatography of the stored samples
were found and no additional peaks appeared when
compared with chromatograms of the freshly prepared
samples. Results are given in Table 6.
Assay of swab samples collected from different loca
tions within the equipment train.
Swab samples from
different locations within the manufacturing equip
ment train were submitted to the laboratory for the
analysis of verapamil hydrochloride residual. These
samples were prepared and analyzed by the proposed
method. For most location (Material dispensing
scoops, Turbo sieve—Bohle, Fluid bed dryer—Glatt
WSG, Washer—extractor Miele, Metal detector—
Lock Met 30+, Tablet press—Kilian) the residues
Table 4.
Linear regression data in the analysis of verapamil
hydrochloride
Statistical parameters Obtained values
Concentration range (
µ
g/mL) 2.50–50.00
Regression equation
y
= 32.598
x
+10.619
Error in slope (
S
b
)0.359
Error in intercept (
S
a
)9.554
Error for
y
est (
S
y
/
x
)16.235
Regression sum of squares (ssreg) 2175488.610
Residual sum of squares (ssresid) 1844.964
F
statistic (
F
) 8254.049
Degrees of freedom (
dF
)7.000
Coefficient of determination (
r
2
) 0.9992
LOD (
µ
g/mL) 1.64
LOQ (
µ
g/mL) 4.98
Table 5.
Precision and accuracy of the results obtained from swabs and plates spiked with verapamil hydrochloride
Sample Amount added,
µ
g/mL Amount found,
µ
g/mL 95% confidence interval, % Recovery, % RSD, % (
n
= 6)
10.00 9.30 91.63–94.29 92.96 1.79
Swabs
20.00 19.08 95.32–95.49 95.41 0.11
30.00 28.93 96.24–96.61 96.42 0.24
10.00 7.86 78.22–78.95 78.59 0.58
Plates
20.00 15.82 78.11–80.11 79.11 1.58
30.00 23.72 78.50–79.63 79.06 0.90
550
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 6 2013
DRAGAN M. MILENOVIC
'
et al.
Tabl e 6 .
Stability results obtained from the verapamil hydrochloride swab extract samples
Sample (
n
= 3)
(
µ
g/swab) Mean recovery
(%) ± CI*; 0 h Mean recovery,
(%) ± CI; 24 h Mean recovery,
% ± CI*; 48 h Mean recovery
(%) ± CI*; 7 days
10.00 93.68 ± 1.05 94.08 ± 1.38 94.11 ± 1.12 93.66 ± 1.67
20.00 93.99 ± 0.49 94.29 ± 0.33 94.00 ± 0.80 93.45 ± 0.92
30.00 97.45 ± 0.26 98.21 ± 0.65 97.73 ± 0.26 97.15 ± 0.18
*Confidence interval.
Table 7.
Results obtained for the determination of verapamil hydrochloride in actual swab samples collected from different
locations within the equipment train
Equipment swabbed Location swabbed* Verapamil hydrochloride detected,
µ
g/swab
Material Dispensing Internal surface <LOQ
Back round plate <LOQ
Scoops External surface <LOQ
Bottom of gran. bowl <LOQ
High Shear Mixer—Diosna Chopper shaft <LOQ
Impeller blade 24.8 (<LSA)
Stainless steel inlet ring <LOQ
Turbo Sieve—Bohle Product inlet, side wall <LOQ
Sieve unit <LOQ
Inside wall <LOQ
Fluid bed Dryer—Glatt WSG Viewing window <LOQ
Bowl bottom mesh <LOQ
Internal surface–top 44.5 (<LSA)
Pillar Hoist—FBD Internal surface–bottom <LOQ
Bowl Inverter Collar <LOQ
Drum back plate <LOQ
Washer—Extractor Drum perforated surface <LOQ
Miele Doormiddle <LOQ
Infeed chute <LOQ
Metal Detector—Lock Reject device, corner <LOQ
Met 30+ Reject flap <LOQ
Inlet plate <LOQ
Deduster—Kramer Dedusting helix 21.0 (<LSA)
Outlet <LOQ
Die table <LOQ
Tablet Press—Kilian Tablet chute cover <LOQ
Main gate <LOQ
* Area swabbed is 25 cm
2
.
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 6 2013
DEVELOPMENT AND VALIDATION OF AN HPLC METHOD 551
were below LOQ. Some of the results obtained for
these samples are presented in Table 7.
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20. International Conference on Harmonization Q2 (R1):
Validation of Analytical Procedures: Text and Method
ology, available at: http://www.ich.org/fileadmin/
Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_
R1/Step4/Q2_R1_Guideline.pdf (05/01/2011).
... Various analytical methodologies were employed to determine VER, MOL, and NHC. Chromatographic methods such as high-performance liquid chromatography (HPLC) [5,[17][18][19][20][21][22] are commonly employed to separate and quantify these compounds. Spectrometric methods, such as UV-Vis spectroscopy [23][24][25], spectrofluorimetry [26,27] contributed significantly to the analysis of these compounds. ...
Article
This study focuses on the determination of the active metabolite of molnupiravir (MOL), N-hydroxycytidine (NHC), using a square wave voltammetric (SWV) method. Carboxylesterase-2 enzymes catalyze the conversion of MOL prodrug into NHC. However, co-administration of verapamil (VER), a carboxylesterase-2 inhibitor, may reduce the levels of NHC, leading to decreasing its antiviral activity. In this context, the levels of NHC and VER were simultaneously monitored using a carbon paste electrode modified with phase I of copper tetracyanoquinodimethane (CuTCNQ) which is highly conductive charge transfer complex. The as-designed sensor was characterized successfully using various spectroscopic techniques and Scanning Electron Microscopy (SEM). The electrochemical behavior of the newly fabricated probe was examined using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). This method demonstrated its efficacy in measuring NHC and VER levels in rabbit plasma samples, showing high sensitivity and selectivity. The calibration plots for the simultaneous quantitation of NHC and VER displayed excellent linearity over the concentration ranges of 50–1600 nmol/L for NHC and 10–250 nmol/L for VER. The limits of detection (LOD) and quantitation (LOQ) in rabbit plasma were found to be 15.2 and 50.8 nmol/L for NHC and 2.9 and 9.9 nmol/L for VER, respectively. Moreover, fundamental pharmacokinetic parameters were calculated for NHC before and after co-administration of VER. The results suggest that the SWV method using CuTCNQ-modified CPE can be a useful tool for the determination of NHC and VER levels in plasma samples, with potential applications in the monitoring of drug-drug interactions involving carboxylesterase-2 inhibitors.
... Следует отметить тенденции унификации анализа в ряду различных объектов при исследовании одного анализируемого вещества: лекарственный препарат, биологические жидкости (кровь, плазма крови, моча), объекты окружающей среды (вода, почвы), другие объекты небиологического происхождения (пищевые продукты, корма) [15,266,386,390,400,413,428,456]. 354,356,448], ВЭЖХ [5,12,29,30,32,44,48,121,124,169,177,231,232,253,257,262,306,337,347,372,379,405,410,456], ВЭЖХ-МС [6,87,234,240,252,268,269,340,349,357,385,391,402,422,452], ГЖХ [102, 107, 179,265,266,270,275,419], ГХ-МС [67, 88, 145, 174, 178, 179, 185, 199, Нами было получено следующее распределение диапазонов определяемых концентраций в крови: ...
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Full-text available
В монографии рассмотрены вопросы разработки методологических основ и экспериментального обоснования подходов судебно-химической экспертизы и фармацевтического анализа к исследованию отдельных многокомпонентных объектов аналитического контроля: биологического материала, сточных вод химико-фармацевтических предприятий, лекарственного растительного сырья, объектов небиологического происхождения. Предложены методологические принципы исследования многокомпонентных объектов в условиях отсутствия стандартных образцов, аттестованных или валидированных методик анализа, аналитического оборудования: принцип использования альтернативных показателей объектов анализа, функционально связанных с содержанием целевого анализируемого вещества и принцип использования альтернативных вариантов количественного определения. Монография предназначена для специалистов в области судебно-химической экспертизы, контроля качества лекарственных средств, а также преподавателей медицинских и фармацевтических вузов, научных работников, аспирантов по специальности 14.04.02 – Фармацевтическая химия, фармакогнозия.
... The drug is used clinically for the treatment of various cardiovascular diseases such as supraventricular tachyarrhythmias, hypertension, nephrotic syndrome, variant angina, cardiomyopathy and all types of ischemic diseases of the heart and vessels [1,3]. To control the quality of verapamil HCl in pharmaceutical preparations, a variety of techniques have been used to analyze it including high performance liquid chromatography [4,5], mass fragmentography [6], spectrophotometry [7], capillary electrophoresis [8], fluorescence [9,10], gas chromatography with mass spectrometry [11], etc. Among these methods, the fluorescence technique presented high sensitivity. ...
Article
Full-text available
An analytical technique based on fluorescence quenching of CdTe/CdS/ZnS quantum dots (QDs) was developed to quantify verapamil in commercially available preparations. Various reaction parameters were optimized and the method developed was validated. One way analysis of variance (ANOVA) and post hoc tests at a 5% significance level were performed to justify the significance of the variation in observations. The linear range of the verapamil concentration was 0.25-5 µg/mL while the limit of detection was 20 µg/mL. Recovery and relative standard deviations were not more than ±10% of the actual amount and <5.9%, respectively. Foreign materials, common metal ions and pharmaceutical excipients of dosage forms caused little interference. To verify the application of the analytical method, the quantity of verapamil in commercially available dosage forms was measured. Verapamil content in the tablets and injections was not more than ±10% of the stated amount and it conformed to the specifications of both the British and the United States pharmacopoeias. In the case of statistical analysis, p-value was <0.05 in almost all levels of all parameters except for the optimized level of system. It can be concluded from the results that the designed method is simple, reliable, cost effective, selective, rapid and sensitive enough to be used for quantitative measurement of the verapamil HCl in dosage forms for quality control purposes.
Article
The analytical performance of sensitive and cost-effective electrochemical sensors based on ionic liquids (ILs) with the bis(trifluoromethylsulfonyl)imide anion, [NTf2]−, and the imidazolium cation with different alkyl chain lengths for electrochemical oxidation of verapamil (VER) was investigated. 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([EMIM][NTf2]), 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][NTf2]) and 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([HMIM][NTf2]) were studied as possible materials for modification of a carbon paste electrode (CPE) for trace-level determination of VER. The experimental parameters including selection of the working electrode, the pH of working media, and the amount of CPE modifiers were investigated. Among them, the [EMIM][NTf2]-CPE with 4.3 wt% of IL was selected as the most appropriate for the square wave voltammetric (SWV) determination of VER at pH 5.0. Cyclic voltammetric studies showed that the electrochemical oxidation of VER was adsorption controlled. Consequently, the square wave adsorptive stripping voltammetric (SW-AdSV) parameters were optimized with Eacc = −0.4 V and tacc = 180 s as the most suitable for accumulation of VER on the electrode surface. The electroanalytical performance of the [EMIM][NTf2]-CPE was further improved by its in situ electrochemical modification with β-cyclodextrin (β-CD) and the linear concentration range of VER was from 0.006 to 0.129 μg mL−1; the relative standard deviation did not exceed 0.7%, and the evaluated limit of detection in model solution was 0.002 μg mL−1. The β-CD/[EMIM][NTf2]-CPE showed adequate selectivity towards VER in the presence of inorganic ions and interferents usually found in human urine. The proposed sensor was successfully applied for VER determination in a spiked human urine sample and pharmaceutical formulation with good repeatability and recovery.
Article
This work presents a simple and low‐cost method for fast and selective determination of Verapamil (VP) in tablets and human urine samples using a boron‐doped diamond working electrode (BDD) coupled to a flow injection analysis system with multiple pulse amperometric detection (FIA‐MPA). The electrochemical behaviour of VP in 0.1 mol L−1 sulfuric acid showed three merged oxidation peaks at around +1.4 V and upon reverse scan, one reduction peak at 0.0 V (vs. Ag/AgCl). The MPA detection was performed applying a sequence of three potential pulses on BDD electrode: (1) at +1.6 V for VP oxidation, (2) at +0.2 V for reduction of the oxidized product and (3) at +0.1 V for cleaning of the working electrode surface. The FIA system was optimized with injection volume of 150 μL and flow rate of 3.5 mL min−1. The method showed a linear range from 0.8 to 40.0 μmol L−1 (R>0.99) with a low limit of detection of 0.16 μmol L−1, good repeatability (RSD<2.2 %; n=10) and sample throughput (45 h−1). Selective determination of VP in urine was performed at+0.2 V due to absence of interference from ascorbic and uric acids in this potential. The addition‐recovery tests in both samples were close to 100 % and the results were similar to an official method.
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The toxicological assessment of medicinal products (MPs) and their residues and metabolites in the environment have become a challenging task worldwide. The contamination of environmental compartments, biota, workplace, foodstuff and feedstuff by residues and metabolites of these substances poses a risk to human health which is still far from being fully understood. On the other hand, existing analytical methods not always possess sufficient detection power to quantify residues of MPs at very low concentrations. This review sets forth some of the most significant contributions made in this field over the past decade with a special focus on novel fit-for-purpose analytical approaches for the detection, identification and quantification of these pollutants and the assessment of their noxious potential for human beings and the environment. Copyright © 2014 Elsevier B.V. All rights reserved.
Article
Verapamil is being used in infants and children but no liquid dosage form is commercially available. The objective of this study was to determine the stability of verapamil in an extemporaneous oral suspension at two temperatures. Commercially available verapamil tablets (80 mg each) were dissolved in purified water and a suspension prepared in 1% methylcellulose and syrup to yield a concentration of 50 mg/ml. The dosage form was stored in ten glass and ten plastic prescription bottles. Half the bottles were stored at 4°C the remainder at 25°C. Two samples were taken from each bottle at 0, 7, 14, 28, 42, 56, 70 and 91 days (n = 10). Verapamil concentrations were measured by a validated and stability-indicating high-performance liquid chromatographic method; the pH was also determined in each sample. The drug was considered stable if its concentration exceeded 90% of the original concentration. In fact, at the end of the study period the mean concentration of verapamil was found to be ≤95% at 4°C and ≤92% at 25°C and was not affected by the type of prescription bottle in which the verapamil had been stored. The pH ranged from 6.7 to 6.8, and did not change at either temperature. Our results indicated that verapamil was stable in an oral suspension for 3 months at both 4°C and 25°C.
Article
A stability indicating HPLC procedure for the determination of verapamil in acidic and basic media is described. The samples were analyzed before and after degradation by HPLC with C-18, CN, and phenyl columns. The peak purity of verapamil in each chromatogram was identified by a Diode Array Detector (DAD). A C-18 column using acetonitrile : triethylamine 7.3 mM pH 3.5 (33:67,v/v) as the mobile phase could be employed in chemical stability studies of verapamil. The LOQ was 2.50 μg/mL and the LOD was 0.833 μg/mL. The degradation of verapamil in 3% H2O2 and 0.1 N NaOH solutions were described. They followed second order kinetics model with rate constants of 498.5 and 339.5 M-1hr-1, respectively. The correlation coefficients were greater than 0.99.
Article
A stability indicating high-performance liquid chromatographic (HPLC) method for determining verapamil hydrochloride in dosage forms is described. The assay affords baseline separation of the drug from its synthesis impurities and from photolytic degradation products, as well as from formulation excipients. The drug was extracted in 0.05 N hydrochloric acid, chromatographed on a C18 reverse-phase column, eluted with methanol-water-acetic acid-triethylamine (55:44:1:0.1) and the effluent was detected at 280 nm. Linearity studies were carried out using peak height or peak area measurements and the detector response to the concentration of verapamil hydrochloride was confirmed. Excellent interlaboratory precision and recovery data were obtained by the spiked placebo method. This procedure was rapid and selective for the assay of the cardiotonic drug. Application of the method for the assay of verapamil hydrochloride in representative dosage forms is described.
Article
The development and validation of an isocratic high performance liquid chromatographic procedure for the determination of trandolapril and verapamil in capsules is reported. The drugs were analysed on a LiChrosorb RP18 column with a mobile phase composed of acetonitrile-methanol-phosphate buffer pH 2.7 (40:40:20) and UV detection at 220 nm. Peak height ratios were linearly related to amounts of the drugs in the range 4–20 μg/mL.The inter-day precision (CV) obtained for the standard solutions ranged from 0.40 to 2.18% for trandolapril and from 0.35 to 2.57% for verapamil. The inter-day coefficients of variation for replicate analyses in capsules ranged from 0.5 to 2.49% for trandolapril and from 0.33 to 1.61% for verapamil.The recovery of analytes after extraction from formulations using the described method, was 99.94 ± 1.69% and 98.13 ± 1.20% (mean ± SD) for trandolapril and verapamil, respectively.
Article
Abstract A high-performance liquid chromatography method for the quantitation of verapamil hydrochloride in pharmaceutical dosage forms has been developed. The method is precise and accurate with a relative standard deviation of 0.63% based on six injections. No preliminary extraction procedure is required to assay injections and a very simple extraction procedure is needed for tablets. There is no interference from the excipients and the method appears to be stability-indicating. The optimum pH range of stability is about 3.2 to 5.6 and the phosphate buffer and ionic strength have very little effect on the stability. Verapamil hydrochloride appears to be a very stable compound since in 105 days at 50°, the aqueous solutions (0.5 mg/ml) did not decompose.
Article
We report a common HPLC method for the single or simultaneous determination of four calcium channel blockers (CCB), namely diltiazem (DTZ), verapamil (VER), nifedipine (NIF) and nitrendipine (NIT) and their active metabolites demetildiltiazem and deacetildiltiazem (MA and M1), norverapamil (NOR), and dehydronifedipine (DHN). DHN was first synthesised in our laboratory and different pH values of the mobil phase were subsequently prepared and tested for chromatographic separation. The detection system and the environmental light conditions were optimised. The best separations of all analytes were obtained using a C(18) column and a mobile phase of methanol, 0.04 M ammonium acetate, acetonitrile and triethylamine (2:2:1:0.04 v/v). Quantitation was performed using imipramine (IMI) as the internal standard. For DTZ and its metabolites (M1 and MA), the wavelength chosen was 237 nm; for VER and its metabolite NOR, it was 210 nm; and, finally for NIF and its metabolite DHN and NIT it was 216 nm. When a simultaneous analysis was carried out the wavelength was of 230 nm. The optimum pH were 7.90 and 7.10 when the separation of NIT and DTZ or VER and NIF were carried out, respectively, and 7.90 when a simultaneous separation was carried out. The detection limit of the assay was less than 8 ng ml(-1) for all compounds, with coefficients of variation less than 7% (for inter- and intra-day) over the concentration range of 1-1000 ng ml(-1). The retention times were less than 11 min. When NIF or NIT were studied, it was necessary to use a sodium vapour lamp in order to avoid the photodegradation which takes place under daylight conditions.
Article
A modification of the USP HPLC method [ United States Pharmacopeia XXII, pp. 1444-1446] for the assay of the purity of verapamil hydrochloride has been evaluated for the determination of the drug content and related compounds in drug raw material. The method enables the resolution of 16 related compounds from the parent drug and, in most cases, from each other. The minimum quantifiable amount for most related compounds is less than 0.05%. Six drug raw material samples are analysed and the total impurities found to be 0.3% or less. All drug assay values were within the USP recommended limits of 99.0-100.5%.
Article
A modified and simple HPLC procedure has been developed for verapamil in plasma. Plasma samples have been vortex-mixed and centrifuged without any need of extraction. The analysis has been performed on a C20 reversed-phase column with fluorometric detection using 5,6-benzoquinoline as an internal standard. Standard curve has been found to be linear for concentrations from 30 to 1000 ng/ml (plasma) for verapamil and no potential source of interference was present. The method has the advantages of speed, small sample requirement and reproducibility. Applicability of the method has been demonstrated by a pharmacokinetic study in 7 rabbits which received a single dose of 0.25 mg verapamil hydrochloride by intravenous administration.
Article
The development of a reversed-phase liquid chromatographic method for the determination of related substances in verapamil hydrochloride is described. The method is based on the use of a simple mobile phase on a specialty base-deactivated reversed-phase column. It enables the resolution of 13 related compounds from the parent drug and from each other. Validation of the method showed it to be reproducible, selective, accurate and linear over the concentration range of analysis with a limit of detection of 0.5 microgram ml-1. The developed method proved to be a real improvement compared with the LC test for chromatographic purity described in the USP monograph for verapamil hydrochloride.