Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 70 No. 2 pp. 301ñ307, 2013
ISSN 0001-6837
Polish Pharmaceutical Society
STABILITY OF CALCIUM FOLINATE (TEVA) IN CONCENTRATE
AFTER RE-USE AND IN DILUTE INFUSIONS IN 0.9% NaCl
IN POLYETHYLENE BAGS
AGNIESZKA KARBOWNIK*, EDYTA SZA£EK, HANNA URJASZ, MALWINA K•DZIO£KA
and EDMUND GRZEåKOWIAK
Department of Clinical Pharmacy and Biopharmacy, Faculty of Pharmacy,
Karol Marcinkowski University of Medical Sciences, åw. Marii Magdaleny 14, 61-861 PoznaÒ, Poland
Abstract: The study concerned the stability of calcium folinate in concentrate in glass vials and diluted in polyethylene (PE) bags stored at 15-25OC and 2-8OC for up to 34 days. Original vials of calcium folinate injection
(10 mg/mL, Teva) were stored at room and refrigerator temperatures and subjected to re-piercing at 1, 2, 3, 7,
14, 22, 28, 30 and 34 days following the initial piercing. Calcium folinate infusions at nominal concentrations
of 0.12 mg/mL were prepared in 0.9% sodium chloride (250 mL) in PE bags. Chemical stability was measured
with a stability-indicating high-performance liquid chromatography (HPLC) assay. Physical stability was
assessed by visual inspection in normal light. The concentration of calcium folinate at each sampling time in
the analyzed solutions remained > 90% of the initial concentration, regardless of the container. No changes in
color or turbidity were observed in any of the vials or in the prepared solutions. Calcium folinate, both undiluted in glass containers and diluted with NaCl 0.9% in PE bags, remains stable (< 10% degradation) for at least
30 days at room and refrigerator temperatures when protected from light.
Key words: calcium folinate, stability, concentrate, infusion
(diluted in 0.9% NaCl, room temperature) (8),
whereas in the generic preparation ñ up to 1 h after
preparation of the solution for infusion (9).
Therefore, it is so important to confirm the stability
of not only the original therapeutic product but also
generic products.
The confirmation of long-term stability
involves the possibility to prepare drugs in
advance for the whole weekend or even for the
whole period of therapy without the involvement
of nursing staff in the process (2). The aim of this
action is to reduce the workload and optimal time
management by pharmacists employed in cytostatic laboratories (1, 3).
Calcium folinate is a folic acid derivative. The
molecular formula of calcium folinate is
C20H21CaN7O7 (M.W. 511.5). Leucovorin is a folic
acid derivative commonly applied in protective
treatment to reduce adverse reactions caused by the
administration of methotrexate and in combination
with 5-fluorouracil in the chemotherapy of advanced
The quality of anti-cancer drugs is inseparably
related with their stability during the storage of originally packaged drugs as well as after opening and
preparation of dilutions. Research on the stability of
cytostatics is especially important due to their low
therapeutic index, serious adverse effects, toxic
degradation products and frequent administration of
maximum therapeutic doses (1). Furthermore, each
drug should be characterized by chemical and physical stability from the moment of preparation to the
end of its administration to the patient (2).
The stability of pharmaceutics is influenced by
numerous factors, such as: temperature, light, solution concentration and pH, solvent and the type of
package where the drug is stored (3). The stability of
each preparation is different, e.g., irinotecan is stable for 24 h (4), oxaliplatin for 14 days (5), panitumumab - for 14 days (6), and vincristine - for as long
as 84 days (7). The stability of a drug may also differ between manufacturers. For example, meropenem in the original preparation is stable up to 6 h
* Corresponding author: e-mail: [email protected]; phone: +48616687854
301
302
AGNIESZKA KARBOWNIK et al.
colorectal cancer, where it intensifies the anti-cancer
effect of the cytostatic (10-13).
The purpose of this study was to determine the
extended chemical and physical stability of calcium
folinate in concentrate in glass vials and diluted in
polyethylene (PE) bags at normal in-use concentration in NaCl.
MATERIALS AND METHODS
Reagents
Calcium folinate, HPLC grade acetonitrile,
ammonium acetate, acetic acid were purchased from
Sigma-Aldrich. Methanol was from Merck. Water
used in the mobile phase was deionized, distilled and
filtered through a Millipore system before use. Vials
containing calcium folinate concentrate 100 mg/10
mL and 1000 mg/100 mL (batch number: 8190110,
1780610) were supplied by Teva Pharmaceuticals
Polska, Warszawa, Poland. The 250 mL polyethylene (ViafloÆ) infusion bags, containing 0.9% sodium chloride (batch number 11B07EON) were purchased from Baxter Polska, Warszawa, Poland.
Chromatographic assay method
The concentration of calcium folinate was carried out by means of a high pressure liquid chromatographic (HPLC) method with UV detection.
The high performance liquid chromatographic
(HPLC) system (Alliance, model 2695; Waters
Associates, Milford, MA, USA) was used with a
diode array detector (DAD) (model 2487; Waters
Associates) and a data acquisition and processing
module (Empower Pro Software 1154; Waters
Associates). Separation was achieved by isocratic
elution of the mobile phase, ammonium acetate 20
mM pH 3.4 (adjusted with acetic acid): acetonitrile
(95 : 5, v/v) at a flow rate of 1.0 mL/min through a
Spherisorb NH2 column (250 mm ◊ 4.6 mm I.D., 5
µm particle size) (Waters). The column temperature
was maintained at 35OC, and the UV-Vis detection
wavelength was set at 267 nm. The samples of 1 µL
were injected into the HPLC system.The total analysis time for each run was 7 min.
Standard preparation
A stock solution of calcium folinate was prepared from accurately weighed (250 mg) pure powder dissolved in water: 0.9% sodium chloride (1 : 1,
v/v, 10 mL). The solution was kept at 4OC. Working
standard solutions were prepared by appropriate
dilutions of the stock solution in 0.9% NaCl to
obtain concentrations across the range of 0.1-15
mg/mL. Quality control (QC) samples were prepared freshly on each day of the experiment.
Calibration curve
The calibration curve was constructed from
plots of peak area versus concentration. The linearity of the method was evaluated at nine calcium folinate concentrations varying from 0.1 mg/mL to 15
mg/mL (three-fold injections).
Intra- and inter-day precision and accuracy
To evaluate the intra- and inter-day accuracy
and precision the QC standard samples at three concentrations were prepared and determined by quantitating five replicates on the same day and on 3
consecutive days. Table 1 shows intra- and interday precision (CV%) and accuracy of this assay
method. The precision of the method at each concentration was calculated as the relative standard
deviation of the mean (CV) by means of the following equation:
CV = (SD/mean) ◊ 100
Accuracy was measured as the percentage difference from the theoretical calculation, according
to the equation:
Table 1. Intra- and inter-day accuracy and precision of calcium folinate (n = 5).
Concentration
(mg/mL)
Mean ± SD
(mg/mL)
Accuracy
(bias %)
Precision
(CV%)
0.5
0.44 ± 0.03
-12.0
6.8
5.0
4.66 ± 0.25
-6.8
5.4
12.5
11.52 ± 0.36
-7.8
3.1
0.5
0.48 ± 0.03
-4.0
6.3
5.0
4.97 ± 0.14
-0.6
2.8
12.5
12.07 ± 0.50
-3.4
4.1
Intra-day
Inter-day
Stability of calcium folinate (TEVA) in concentrate after reuse...
Bias (%) = (concentrationmeasured ñ concentrationtheoretical)/
( concentrationtheoretical) ◊ 100%
Limit of detection (LOD) and lower limit of
quantification (LLOQ)
The LOD was defined as the analyte concentration giving a signal to noise ratio of 3 : 1. The
LLOQ was defined as the analyte concentration giving a signal to noise ratio of 10 : 1. Under the optimized conditions, the LOD and LLOQ of the calcium folinate concentrations were 100 ng/mL and 0.1
mg/mL, respectively.
Preparation of calcium folinate infusion
All calcium folinate infusions were prepared
under EU Class A conditions, in accordance with
the principles of Good Pharmaceutical Manufacturing Practice. Calcium folinate infusions at
nominal concentrations of 0.12 mg/mL, were prepared in 0.9% sodium chloride (250 mL) in PE bags.
Twelve of the infusions were stored, well protected
from light in green polythene overwraps, six at room
temperature (19.1 ± 0.3OC) and six at refrigerator
temperature (3.7 ± 1.3OC). The samples were analyzed immediately after preparation (t = 0) and at the
following scheduled time intervals 1, 2, 3, 7, 14, 22,
28, 30, 34 days. The concentrations of the calcium
folinate in the analyzed samples were calculated by
means of the regression equation of the straight line
y = ax + b for lower and higher concentration.
Visual inspection
Infusions were visually inspected under standard laboratory lighting against dark and light backgrounds for changes in clarity, color, and presence
of particulate matter.
303
RESULTS AND DISCUSSION
The calibration curve of the peak area versus
concentration was found to be linear over the evaluated range of 0.1-15 mg/mL in 0.9% NaCl. The calibration equation was:
y = 1.47 ◊ 106x + 4.52 ◊ 104
The linear regression coefficient in plasma
was r2 = 0.999 and linearity was achieved in this
range (Fig. 1). Intra- and inter-day precision and
accuracy of the LQC (0.5 mg/mL), MQC (5
mg/mL), and HQC (12.5 mg/mL) were well within
the acceptable limit of 15% coefficient of variation
(CV%). The results of intra-day precision and accuracy (n = 5) are summarized in Table 1. Intra-day
precision and inter-day precision were acceptable
with all CVs less than 6.8%. The intra-day accuracy and inter-day accuracy were also acceptable with
the range of 88.0-99.4%. Figure 2 A-D shows typical chromatograms obtained from a drug-free solution and a solution of calcium folinate in 0.9%
NaCl, respectively. The chromatograms show that
the separation from matrix constituents is sufficient
for reliable quantitation and no endogenous components interfered with the analyte peak. Calcium folinate peak was detected with retention time 3.219 ±
0.171 min.
Drug stability is the subject of interest of many
researchers. They proved that sunlight strongly
affects the stability of leucovorin. The stability of
calcium folinate is also significantly influenced by
pH and high temperature. After preparation of solutions with extreme pH values: alkaline (pH = 12.34)
and acidic (pH = 1.26) and heating them for 60 min
to the temperature of 100OC the products of leucovorin degradation were observed (14).
Figure 1. Calibration curves of calcium folinate within the concentration range of 0.1-15 mg/mL (n = 5)
304
AGNIESZKA KARBOWNIK et al.
Figure 2. The chromatogram of calcium folinate: a drug-free solution (A), solution of calcium folinate in 0.9% NaCl, day 0 ñ 1.308 mg/mL
(B), day 30 ñ 1.285 mg/mL (C), day 34 ñ 1.102 mg/mL (D)
Lecompte et al. (2) proved 4-day stability of
calcium folinate in the form of lyophilized powder
after dilution in a solution of 0.9% NaCl and 5%
glucose for the concentrations of 1.0 and 1.5 mg/mL
stored both in PVC bags and in glass bottles at the
temperature of 4OC and 23OC without access to light.
On the other hand, the solutions with the concentrations of 0.1 mg/mL in 0.9% NaCl stored both in
PVC bags and in glass bottles at the temperature of
4OC and 23OC and 0.5 mg/mL in 0.9% NaCl stored
both in PVC bags and in glass bottles at the temperature of 4OC and 23OC were proved to be unstable
(2). Cadrobbi et al. (15) proved 30-day stability of
sodium folinate. The solutions under analysis were
diluted in 5% glucose (250 mL) at the concentration
of 3.2 mg/mL and were stored in polyolefin bags at
the temperature of 4OC (15).
Lebitasy et al. (14) conducted research on the
influence of freezing and defrosting on the longterm stability of the active form of calcium folinate.
In order to do so they prepared drug dilutions in 250
mL of 5% glucose and obtained the final active substance concentration of 1.60 mg/mL in polyolefin
infusion bags. The samples under investigation were
frozen at a temperature of -20OC for 95 days and
then they were defrosted in a microwave and stored
at a temperature of 5 ± 3OC for a month. The investigation proved the stability of calcium folinate both
after defrosting of the samples and in a 30-day storage period (100.60 ± 0.96%) in reference to the initial values of the concentrations measured immediately after preparation of the dilutions. Physical stability was also evaluated. The team of researchers
did not observe changes in color, the appearance of
precipitate in the samples immediately after defrosting and during the 30 days of storage (14).
The authorsí own research consisted in analysis
of the drug at the concentration of 10 mg/mL and the
volumes of 10 and 100 mL after opening of an original glass vial, stored at room temperature (19.1 ±
0.3OC) and at refrigerator temperature (3.7 ± 13OC)
without access of light. The concentration was measured immediately on opening the preparation and
after 24, 48, 72 h, 7, 14, 22, 28, 30 and 34 days. After
30 days, the mean percentage variation in the concentration of leucovorin was: 100.18 ± 0.13% for
Leucovorin Ca Teva concentrate 1000 mg/100 mL at
the temperature of 3.7 ± 1.2OC (Tab. 4), 100.92 ±
305
Stability of calcium folinate (TEVA) in concentrate after reuse...
Table 2. Calcium folinate (%) at different sampling times in concentrate, stored at refrigerator temperature (2-8OC) and room temperature
(15-25OC) for Leucovorin Ca TevaÆ 100 mg/10 mL.
Percentage of initial concentration at indicated time (day)
Temp.
0
1
2
3
7
14
22
28
30
34
2-8OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.21
0.14
3
pass
100.00
0.09
3
pass
99.99
0.15
3
pass
100.04
0.42
3
pass
100.09
0.06
3
pass
100.10
0.22
3
pass
100.09
0.17
3
pass
100.12
0.09
3
pass
86.96
5.32
3
pass
15-25OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
99.92
0.29
3
pass
99.80
0.56
3
pass
99.89
0.41
3
pass
100.03
0.08
3
pass
100.09
0.06
3
pass
99.98
0.07
3
pass
100.04
0.14
3
pass
100.00
0.12
3
pass
88.38
8.29
3
pass
Table 3. Calcium folinate (%) at different sampling times in sodium chloride 0.9%, stored at refrigerator temperature (2-8OC) and room
temperature (15-25OC) for Leucovorin Ca TevaÆ 100 mg/10 mL.
Percentage of initial concentration at indicated time (day)
Temp.
0
1
2
3
7
14
22
28
30
34
2-8 C
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.28
0.16
3
pass
99.37
0.37
3
pass
99.19
1.14
3
pass
100.10
0.36
3
pass
98.06
1.68
3
pass
98.06
2.46
3
pass
96.54
1.63
3
pass
97.92
1.34
3
pass
87.42
13.12
3
pass
15-25OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.58
1.78
3
pass
100.85
0.33
3
pass
98.48
0.39
3
pass
97.82
2.25
3
pass
100.57
0.44
3
pass
98.60
1.61
3
pass
98.69
2.07
3
pass
99.47
1.43
3
pass
89.92
8.93
3
pass
O
Table 4. Calcium folinate (%) at different sampling times in concentrate, stored at refrigerator temperature (2-8∞C) and room temperature
(15-25∞C) for Leucovorin Ca TevaÆ 1000 mg/100 mL..
Percentage of initial concentration at indicated time (day)
Temp.
0
1
2
3
7
14
22
28
30
34
2-8OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.10
0.08
3
pass
100.13
0.05
3
pass
100.09
0.05
3
pass
100.13
0.03
3
pass
100.01
0.05
3
pass
100.11
0.12
3
pass
100.08
0.07
3
pass
100.18
0.13
3
pass
80.45
3.90
3
pass
15-25OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.15
0.19
3
pass
100.25
0.19
3
pass
100.09
0.31
3
pass
100.20
0.20
3
pass
100.20
0.13
3
pass
100.62
0.12
3
pass
99.04
0.68
3
pass
100.11
0.32
3
pass
84.03
4.16
3
pass
306
AGNIESZKA KARBOWNIK et al.
Table 5. Calcium folinate (%) at different sampling times in sodium chloride 0.9%, stored at refrigerator temperature (2-8OC) and room
temperature (15-25OC) for Leucovorin Ca TevaÆ 1000 mg/100 mL.
Percentage of initial concentration at indicated time (day)
Temp.
0
1
2
3
7
14
22
28
30
34
2-8OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.25
1.75
3
pass
98.81
2.86
3
pass
100.21
0.77
3
pass
100.30
1.74
3
pass
100.08
0.74
3
pass
100.78
1.16
3
pass
99.90
0.79
3
pass
100.92
1.47
3
pass
84.10
13.69
3
pass
15-25OC
Mean
SD
n
Visual
apperance
100.0
0.0
3
pass
100.95
2.12
3
pass
101.82
2.37
3
pass
100.83
0.79
3
pass
101.98
2.29
3
pass
101.15
1.97
3
pass
100.99
0.67
3
pass
100.86
1.69
3
pass
100.60
1.67
3
pass
82.84
4.69
3
pass
1.47% diluted in 0.9% NaCl Leucovorin Ca Teva
1000 mg/100 mL at the temperature of 3.7 ± 1.2OC
(Tab. 5), 100.11 ± 0.32% for Leucovorin Ca Teva
concentrate 1000 mg/100 mL at the temperature of
19.1 ± 0.3OC (Tab. 4), 100.60 ± 1.67% diluted in
0.9% NaCl Leucovorin Ca Teva 1000 mg/100 mL at
the temperature of 19.1 ± 0.3OC (Tab. 5), 100.12 ±
0.09% for Leucovorin Ca Teva concentrate 100
mg/10 mL at the temperature of 3.7 ± 1.2OC (Tab. 2),
97.92 ± 1.34% diluted in 0.9% NaCl Leucovorin Ca
Teva 100 mg/10 mL at the temperature of 3.7 ± 1.2OC
(Tab. 3), 100.00 ± 0.12% for Leucovorin Ca Teva
concentrate100 mg/10 mL at the temperature of 19.1
± 0.3OC (Tab. 2), 99.47 ± 1.43% diluted in 0.9 %
NaCl Leucovorin Ca Teva 100 mg/10 mL at the temperature of 19.1 ± 0.3OC (Tab. 3).
On the other hand, after 34 days, the mean percentage variation in the concentration of leucovorin
was: 80.45 ± 3.90% for Leucovorin Ca Teva concentrate 1000 mg/100 mL at the temperature of 3.7
± 1.2OC (Tab. 4), 84.10 ± 13.69% diluted in 0.9%
NaCl Leucovorin Ca Teva 1000 mg/100 mL at the
temperature of 3.7 ± 1.2OC (Tab. 5), 84.03 ± 4.16%
for Leucovorin Ca Teva concentrate 1000 mg/100
mL at the temperature of 19.1 ± 0.3OC (Tab. 4),
82.84 ± 4.69% diluted in 0.9% NaCl Leucovorin Ca
Teva 1000 mg/100 mL at the temperature of 19.1 ±
0.3OC (Tab. 5), 86.96 ± 5.32 for Leucovorin Ca Teva
concentrate 100 mg/10 mL at the temperature of 3.7
± 1.2OC (Tab. 2), 87.42 ± 13.12% diluted in 0.9%
NaCl Leucovorin Ca Teva 100 mg/10 mL at the temperature of 3.7 ± 1.2OC (Tab. 3), 88.38 ± 8.29% for
Leucovorin Ca Teva concentrate 100 mg/10 mL at
the temperature of 19.1 ± 0.3OC (Tab. 2), 89.92 ±
8.93% diluted in 0.9% NaCl Leucovorin Ca Teva
100 mg/10 ml at the temperature of 19.1 ± 0.3OC
(Tab. 3).
Having considered the temperature of storage
of the solutions under investigation and the results
of the experiment no correlation between the stability of leucovorin and refrigerator and room temperatures was observed.
During the entire period of storage no changes
in color, turbidity or precipitation were observed in
any of the leucovorin solutions under investigation.
The protection of the drug from access to light was
necessary due to the indications of the manufacturer, who proved photodegradation of leucovorin in
licensing investigations.
CONCLUSION
Calcium folinate appears to be physically and
chemically stable for at least 30 days in concentrate
in glass containers or diluted with 0.9% sodium
chloride in PE bags at a concentration of 0.12
mg/mL, at refrigerator temperature (2-8OC) and
room temperature (15-25OC) when protected from
light.
After 34 days of storage of open leucovorin
concentrates and dilutions in 0.9% NaCl solution the
mean concentration of the active substance was
under 90%, which proves the decomposition of calcium folinate and the presence of degradation products. This fact is confirmed by extra chromatographic peaks.
Stability of calcium folinate (TEVA) in concentrate after reuse...
Acknowledgment
This study was supported with an educational
grant from Teva Poland.
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Received:19. 06. 2012