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ISSN 10619348, 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(3,4dimethoxyphenethyl)
N
methylamino]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.
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
“ZdravljeActavis” 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 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
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, 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
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 RP18e, 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
solutesilanol 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 polivinylplastics) 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
Polivinylplastics 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 autosampler 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 Doormiddle <LOQ
Infeed 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.
REFERENCES
1. Guide to Inspections Validation of Cleaning Processes,
U.S. Food and Drug Administration, available at: http://
www.fda.gov/ICECI/Inspections/InspectionGuides/
ucm074922.htm (05/01/2011).
2. Cleaning Validation Guidance, Guidance on aspects of
cleaning validation in active pharmaceutical ingredient
plants, Dec. 2000, available at: http://apic.cefic.org/
pub/pubcleaningvalidation.pdf (05/01/2011).
3. Guide to Cleaning Validation in API plants, Cleaning
Validation in Active pharmaceutical ingredient manu
facturing plants, Sep. 1999, available at: http://apic.cefic.
org/pub/4CleaningVal9909.pdf (05/01/2011).
4. World Healthy Organization, Supplementary guide
lines on good manufacturing practices (GMP): Valida
tion, Working document QAS/03.055/Rev.2, pp. 24
⎯
33,
available at: http://www.who.int/medicines/services/
expertcommittees/pharmprep/Validation_QAS_055_
Rev2combined.pdf (05/01/2011).
5. Ozkan, Y., Yilmaz, N., Ozkan, A.S., and Biryol, I.,
Il
Farmaco
, 2000, vol. 55, no. 5, p. 376.
6. Garcia, A.M., Solans, C., Aramayona, J.J., Fraile, J.L.,
Bregante, A.M., and Castillo, R.J.,
Tal an ta
, 1998,
vol. 47, no. 5, p. 1245.
7. Venkatesh, G., Ramanathan, S., Mansor, M.S.,
Nair, K.N., Sattar, A.M., Croft, L.S., and Navaratnam, V.,
J. Pharm. Biomed. Anal.
, 2007, vol. 43, no. 4, p. 1546.
8. Valvo, L., Alimenti, R., Alimonti, S., Raimondi, S.,
Foglietta, F., and Campana, F.,
J. Pharm. Biomed.
Anal.
, 1997, vol. 15, no. 7, p. 989.
9. Lacroix, M.P., Graham, J.S., and Lovering, G.E.,
J. Pharm. Biomed. Anal.
, 1991, vol. 9, no. 10–12,
p. 817.
10. He, L. and Wang, S.,
Arch. Pharm. Res.
, 2003, vol. 26,
no. 9, p. 763.
11. Chen, L.G., Li, F.Y., and Lee, Y.F.,
Chin. Pharmaceut.
J.
, 2000, vol. 52, no. 2, p. 113.
12. Gupta, D.V.,
Drug Develop. Ind. Pharm.
, 1985, vol. 11,
no. 8, p. 1497.
13. Nahata, C.M.,
J. Appl. Therap.
, 1997, vol. 1, no. 3,
p. 271.
14. Gumieniczek, A. and Hopkala, H.,
J. Liq. Chrom.
R. T.
, 2001, vol. 24, no. 3, p. 393.
15. Kasturi, K., Rao, S.D., Sundaram, R., and Pharma, T.,
Ind. Drugs
, 1984, vol. 21, no. 10, p. 463.
16. Tsilifonis, C.D., Wilk, K., Reisch, R., and Daly, E.R.,
J. Liq. Chrom.
, 1985, vol. 8, no. 3, p. 499.
17. Yazan, Y. and Bozan, B.,
Pharmazie
, 1995, vol. 50,
no. 2, p. 117.
18. ElGhany, M.F.A., Moustafa, A.A., Elzeany, E.B., and
Stewart, T.J.,
J. Plan. Chromatogr. Mod. TLC
, 1996,
vol. 9, no. 5, p. 388.
19. Eiden, F. and Karin, G.B.,
Pharm. Zeit.
, 1984, vol. 129,
no. 12, p. 678.
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).