Report 5-FU 35 Days ENG 150115

N10566-A
BIOFORDRUG srl
Spin-off dellUniversit degli Studi di Bari A. Moro - BARI -
Via Orabona 4, 70125, Bari, Italy (c/o Dipartimento di Farmacia-Scienze del Farmaco)
PI: 07162850726;
Tel: +39-0805442727; Fax +39-0805442231;
website: www.biofordrug.com; e-mail: nicolaantonio.colabufo@uniba.it; info@biofordrug.com
REPORT N 1/2014
Object
Stability Study of Fluorouracil in MyFuser M2

Elastomeric Infusion Pump Manufactured by Canox
Authors
Biofordrug s.r.l. - Spin-off Universit degli Studi di Bari
INTRODUCTION
The purpose of this report is to study the stability of Fluorouracil (5-FU) into the
elastomeric infusion pump MyFuser M2 in Normal Saline, at storage periods and
temperatures similar to those the drug is usually prepared and stored in Pharmacies and
Hospitals for administration to patients. In particular, Fluorouracil 25 mg/mL in normal
saline has been studied in the elastomeric pump. The study was performed at 25C and
4C.
The part of device that is on contact to the drug is the elastomeric membrane and the
infusion plastic line at the moment of the sampling.
As reported before, the employed dose was 25 mg/mL that is in accordance with the
dosage used in infusion therapy.
Biofordrug srl is certified ISO9001-2008 Reg. 10566-A for carrying out the studies of
drug stability in medical devices for clinical employment.

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SUMMARY
I. PROTOCOL
1. Test product
2. Filling procedure
3. Medical device
4. Storage temperature
5. Preparation of test samples and sampling
6. Sample Collection
7. HPLC Apparatus
8. LC/MS Apparatus
9. Robustness
II. RESULTS
III. CONCLUSIONS
IV. EXPERIMENTAL SECTION

1. Chemicals and Reagents
2. HPLC apparatus
3. HPLC Operating Conditions
4. Test sample preparation and testing frequency
5. Chromatograms
6. LC-MS analyses
I. PROTOCOL

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1. Test product. The employed drug is described in the Table 1
Drug
Volume
Filled
Concentration
Diluent
Brand
Manufacturer
Batch
Expiry
date
Fluorouracil
100 mL
25 mg /mL
NS
FLUOROURACILE
TEVA 250 mg/5 ml
Teva Italia S.r.l.
011083
02/2016
Table 1
2. Filling Procedure
The tested concentration (25 mg/mL) was prepared diluting 50 ml of Fluorouracil (250
mg/5 mL) with 50 mL of Normal Saline. Then the prepared solution was poured into the
elastomeric pump. The filling procedure of the elastomeric pumps was executed according
with protocols of the Unit of Antineoplastic Drug Handling (U.Ma.CA.) at the Cancer
Institute "Giovanni Paolo II", Bari. The manipulation was performed in accordance with
international standard guidelines on Injectables. The number of filled elastomers is
reported in the Table 2.
Concentration
25 mg/mL
Temperature
25C
4C
N of Devices
3. Medical device. MyFuser M2; LOT G12F172; Dispensing speed: 2.0 mL/h;
orage temperature. Elastomeric pumps have been stored at 25C and 4C as reported in
Table 3.

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4. Storage temperature. Elastomeric pumps have been stored at 25C and 4C as

reported in Table 3.
Concentration
25 mg/mL
Diluent
NS
Storage
Condition
N of Devices
25C
4C
Table 2
In particular, the study at 25C have been stored performed in an electric oven bearing the
thermostat. The samples for analysis at 4C have been stored in a laboratory refrigerator
5. Preparation of test samples and sampling. Samples were taken directly from the
elastomeric pump (2 mL x 2) immediately after filling (T=0) and at subsequent days
indicated in the Table 4.
Concentration
25 mg/mL

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Diluent
Storage
Condition
Determinations*
NS
25C
4C
4, 7, 14, 23, 30, 35 d
4, 7, 14, 23, 30, 35 d
Table 4 (* d=day)
6. Sample Collection. The collection was carried out directly by the terminal part of the
plastic infusion line in a glass tube and then the solution was transferred into vials for
HPLC and LC-MS analyses. The taken volume was 2 mL for each analysis. In total, the
taken volume was 4 mL for each requested determination. The samples were used as
such in HPLC and LC-MS Analyses.
7. HPLC Apparatus. HPLC Analysis were performed on an Agilent 1260 Infinity
instrument equipped with 1260 DAD VL + detector controlled by OpenLAB CDS
ChemStation Edition Software. The employed column was an Agilent Poroshell 120 ECC18 (3.0 x 50 mm, 2.7 m) column. HPLC operating conditions (Wavelength, Flow, Mobile
phase, Injection volume, Retention time, Column temperature) are reported in
Experimental Section.
8. LC/MS Apparatus. LC-MS analyses were performed on an Agilent 1100 LC/MSD trap
system V spectrometer equipped with an electrospray ionization (ESI-MSn) system. The
samples (2 mL) were analysed after Lyophilization and subsequent extraction with an
appropriate solvent (AcOEt).

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9. Robustness.
The precision of the method was verified by repeatability in a single day and the
intermediate precision by different analysts on different days. Repeatability and
intermediate precision were maintained by analysing the samples in triplicate. The results
are summarized for each drug and the corresponding typical chromatograms have been
reported. Moreover the critical parameters of flow rate and temperature were changed to
assess the method performance.

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II. RESULTS
In this study the employed method involved the use of a combined HPLC-LCMS approach.
The samples of loaded solution were taken over the studied period and submitted to HPLC
analysis and LC-MS evaluation allowing an unambiguous assessment of drug purity,
stability and compatibility.
The used procedure for establishing purity and stability of each tested drug was to
compare the area of peaks under interest with that of a reference solution. In addition, it is
important to evaluate the area of the peaks because this parameter could be employed to
appreciate the stability of drug concentration during the period of analysis. If fact, the
compound could be chemically stable but into the pump could arise a gradient of
concentration. Then the percentage changes of peak area were evaluated over all
indicated period. In particular % changes of areas, reported in the Table 5, were
calculated according to the following formula:
(Tx-T0/T0) 100
in which T0 is the percentage of peak area corresponding of tested compound immediately
subsequent to the filling of the elastomeric pump and Tx is the percentage area at different
days. The T0 usually compared with the standard sample having the same concentration of
filled solution. Final reported values of % change in areas are the average value between
two different determinations related to two different elastomers.
The acceptance criteria in solution concentration are established by considering the
clinical commonly used dose.
Moreover, particular attention was given to the monitoring of colour and clarity of the
solution into the elastomer and into the taken samples. In this regard, there are no findings
to communicate.

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Fluorouracil in NS
T
Area (%)
% Change in Areas
100
25 C
4d
100
25 C
7d
99.99
0.01
25 C
14 d
100.04
0.04
25 C
23 d
99.92
0.08
25 C
30 d
99.98
0.02
25 C
35 d
99.96
0.04
4 C
4d
100.01
0.01
4 C
7d
100.02
0.02
4 C
14 d
100
4 C
23 d
99.98
0.02
4 C
30 d
99.97
0.03
4 C
35 d
99.97
0.03
Table 5

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I.
CONCLUSIONS
Qualitative
The obtained HPLC-chromatograms and LC-MS analyses of reference solution and of all
samples displayed that Fluorouracil is stable in the medical device for the experimental
conditions reported in Table 4. In fact, as reported in HPLC and LC-MS studies, additional
peaks due to degradation of drug and/or to medical device-drug interaction, are not
detected
Quantitative
The study of % variation of areas has been employed to appreciate the stability of drug
concentration during the requested period of observation. As reported in the Table 5, very
low variability during the studied period and at all experimental conditions has been
observed. The findings allow to confirm that, for each determination, the drug
concentration is constant and superimposed with respect to reference solution and the
sample at T=0 and in accordance with the common employed concentration.

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IV. EXPERIMENTAL SECTION

1. CHEMICALS AND REAGENTS. All chemicals and reagents were of the highest purity.
All solvents were HPLC grade quality and all chemicals were purchased from SigmaAldrich Co. (St. Louis, USA).
2. HPLC APPARATUS
HPLC: Agilent 1260 infinity- Software Openlab CDS
Column: Agilent Poroshell 120 EC-C18 3.0 x 50 mm, 2.7 m.
Detector: DAD
2. HPLC Operating Conditions

Wavelength: 254 nm
Flow: 0.1 mL/min
Mobile phase: (Acetonitrile: Water) = (30:70)
Injection volume: 0.1 L
Retention time: about 2.5 min
Column temperature: 40C
3. Test sample preparation and testing frequency

The samples, taken on the indicated days, were used as such by HPLC.

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4. Chromatograms
Examples of chromatograms obtained by HPLC analysis of taken samples are reported
below.
a) T = 0 in NS
b) T = 4 d at 25C in NS
c) T = 7 d at 25C in NS
d) T = 14 d at 25C in NS

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e) T = 21 d at 25C in NS
f) T = 30 d at 25C in NS
g) T = 35 d at 25C in NS
h) T = 4 d at 4C in NS

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i) T = 7 d at 4C in NS
l) T = 14 d at 4C in NS
m) T = 21 d at 4C in NS
n) T = 30 d at 4C in NS

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o) T = 35 d at 4C in NS
5. LC-MS Analyses
An example of ESI-negative analyses of samples taken at each determination are reported
below.
5-Fluorouracil (MW = 130 g/mol): ESI-/MS m/z: 129 [M-H]-. ESI-/MS/MS m/z:113 (100).
Bari, January 15, 2015

Report 5-FU 35 Days ENG 150115

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N10566-A

Stability Study of Fluorouracil in MyFuser M2

Biofordrug s.r.l. - Spin-off Universit degli Studi di Bari

IV. EXPERIMENTAL SECTION

1. Test product. The employed drug is described in the Table 1

Teva Italia S.r.l.

4. Storage temperature. Elastomeric pumps have been stored at 25C and 4C as

4, 7, 14, 23, 30, 35 d

4, 7, 14, 23, 30, 35 d

IV. EXPERIMENTAL SECTION

2. HPLC Operating Conditions

3. Test sample preparation and testing frequency

Bari, January 15, 2015

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