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org on December 15, 2016

Chemical Stability of Two Sterile, Parenteral Formulations of

Cyclophosphamide (Endoxan ®) after Reconstitution and Dilution
in Commonly used Infusion Fluids
J. H. Beijnen, R. van Gijn, E. E. Challa, et al.

PDA J Pharm Sci and Tech 1992, 46 111-116

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RESEARCH ARTICLE

Chemical Stability of Two Sterile, Parenteral Formulations of

Cyclophosphamide (Endoxan®) after Reconstitution and
Dilution in Commonly used Infusion Fluids
J. H. BEIJNEN*-, R. van GUN*, E. E. CHALLA*, G. P. KAIJSER* and W. J. M. UNDERBERGt

"Slotervaart Hospital I Netherlands Cancer Institute, Amsterdam, and fDepartment of Pharmaceutical Analysis,
Faculty of Pharmacy, State University of Utrecht, Utrecht, The Netherlands

ABSTRACT: The commercially available parenteral dosage forms of cyclophosphamide (Endoxan®, Cycloblas-
tine®) are manufactured by an aseptic dry-filling technique and exhibit a slow dissolution rate. A novel dosage
form has been developed bv one of the manufacturers based on the technique of freeze drying. Dissolution rates of
both types of formulations were determined and it was shown that the freeze-dried formulation dissolves more
rapidly, within 20 seconds, while it takes at ¡east three minutes to dissolve the dry-filled formulation. The chemical
stabilities of the cyclophosphamide solutions, obtained after reconstitution and lor dilution of both formulations,
have been investigated and tested as a function of drug concentration (20 and I mglmL). solvent (water. 0.9%
sodium chloride, 5% dextrose), container material (glass and polyvinyl chloride (pvc)), light conditions (normal
room fluorescent light /dark) and temperature (4°, 20-22° and 37°C). The test solutions were analyzed by a
stability-indicating reverse phase high performance liquid chromatographic method with ultraviolet detection at
214 nm. Cyclophosphamide solutions (solvent: water; drug concentration: 20 mgimL) are stable when stored for
seven days at 4°C in the dark. At higher temperatures degradation occurred during the test period with 10% loss
afterseven days at ambient temperature and 50% loss after seven days storage at 3TC. Similar data were found in
admixtures with 5% dextrose and 0- 9% sodium chloride and initial drug concentration ofl mglmL. There are no
significant differences in chemical stability between the solutions obtained from reconstitution and dilution of the
dry-filled and lyophilized formulations. The type of container materials tested (glass and pvc) have no influence
on the overall chemical stability of cyclophosphamide (concentration: 20 mglmL) in the dark On the other hand,
in diluted solutions (drug concentration: 1 mglmL in 0.9% sodium chloride) exposed to normal room fluorescent
light in a day-night cycle at ambient temperature, cyclophosphamide is stable in pvc (less than 5% degradation
after seven days storage) but shows 10% degradation in glass. In PVC minibags containing 0.9% sodium chloride,
cyclophosphamide (1 mglmL) shows >5% degradation when stored for seven days at ambient temperature in
the dark. When exposed to normal fluorescent light at ambient temperature the drug (1 mglmL) degrades for
approximate 5% in both glass and PVC containers.

introduction of the drug in the liver leads to the formation of

Cyclophosphamide (2-[bis(2-chloroethyl)amino]-tet-
rahydro-2H-l,3,2-oxazaphosphorine 2-oxide; Endoxan®,
Cytoxan*. Neosar®, Cycloblastine®) is one of the most
frequently used alkylating agents in cancer chemother-
apy. The drug was first synthesized in 1957 (1) and is
mainly used in combination chemotherapy, for example
with methotrexate and 5-fluoro-uracil for the treatment
of breast cancer and in combination with etoposide and
doxorubicin for small cell lung cancer. Cyclophospha-
mide itself, like a pro-drug, has no biological activity and
requires in vivo activation, occurring through a multistep
process to yield the cytotoxic active species. Activation
Received November 4, 1991. Accepted for publication February 20.
1992.
- Author to whom correspondence is to be addressed: Dr. J. H.
Beijnen. Slotervaart Hospital/Netherlands Cancer Institute. Lou-
wesweg 6, 1066 EC Amsterdam, The Netherlands.
4-hydroxy-cyclophosphamide which is in equilibrium
with its tautomer aidophosphamide. Subsequent sponta-
neous ^-elimination generates acrolein and phosphor-
amide mustard from this intermediate. Urinary excre-
tion of acrolein has been implicated in the cystitis caused
by cyclophosphamide while phosphoramide mustard is
considered as the primary active, alkylating species.
Other metabolic routes have also been identified but are
probably of less importance (2).
Cyclophosphamide has been marketed for over 25
years in Europe in tablet form and as a sterile powder
for injection. The latter is a mixture of cyclophospha-
mide (monohydrate) and sodium chloride. This intrave-
nous (IV) formulation is manufactured by an aseptic
dry-filling technique. Sterile Water for Injection must be
added in preparing the solution for IV administration.
Unfortunately, the powder dissolves slowly and only
with vigorous agitation. This is time consuming and

Vol. 46, No. 4 / July-August 1992 111

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holds a potential risk for breakage of the vials. In our phamide using high performance liquid chromatography
practice this is considered an important disadvantage in (HPLC). The solutions were also visually inspected for
the use of this cyclophosphamide formulation. The clarity, color and signs of precipitation using the light
dissolution rate of the drug can be accelerated by briefly box. All admixtures were prepared under aseptic condi-
heating the solution, however this can lead to degrada- tions in a vertical laminar-airflow biological safety cabi-
tion of the drug. Therefore, this procedure is not net (Model DLF/BSS6. Clean Air Company. Woerden.
recommended (3). The dissolution can be speeded up by The Netherlands).
the addition of a minor quantity of ethanol followed by The liquid chromatograph consisted of a Model 6000
dilution with water. This technique is dissuaded as A solvent delivery system (Waters Assoc. Milford. MA.
ethanol can cause local irritation during IV administra- USA) and an automatic sample injection device model
tion. The manufacturer of cyclophosphamide (En- SP8880 (Spectra Physics. Santa Clara. CA. USA). Sepa-
doxan\ Asta Pharma AG. Frankfurt am Main. Ger- ration was performed on a Lichrosorb RP-18 (particle
many) has. therefore, put research efforts into the size: 5 |xm) column (125 mm x 4 mm internal diameter:
development of an alternative formulation based on the Merck. Darmstadt. Germany). The mobile phase con-
freeze-drying process. A lyophilized product will usually sisted of 200 mL acetonitrile and 800 mL 5 mM
constitute more rapidly and completely compared to a phosphate buffer pH 6.5. The flow rate was maintained
dry-filled product. Furthermore, in view of current at 1.0 mL/minute. The column effluent was monitored
concern over potential risks from low level exposure to a spectrophotometrically using a Model 441 UV detector
cytotoxic drug during the manufacturing process, the (Waters Assoc.) equipped with a zinc lamp and a fixed
dry-filling technique is not preferable. The purpose of wavelength filter of 214 nm. Retention times and peak
this study was to determine the dissolution rates of areas were measured with an SP-4290 (Spectra Physics)
different formulations of cyclophosphamide and the data system.
chemical stability of the drug after reconstitution and
dilution for IV chemotherapy. The choice of the solvents
Results and Discussion
and conditions are based on current pharmacy practice.
The poor dissolution rate of the dry-filled formula-
Materials and Methods tions of cyclophosphamide has induced the use of
heating and cosolvents to speed up the reconstitution
The commercially available, pharmaceutical dosage step. These techniques, however, should not be encour-
forms of cyclophosphamide containing 0.5 g of the drug aged because of degradation of the drug during heating.
per vial (Endoxan\ Lot 900410, Asta Pharma AG.
Frankfurt am Main. Germany and CycIoblastine\ Lot
90114, Farmitalia Carlo Erba. Milano, Italy) and the
lyophilized formulation of cyclophosphamide (1 g; En-
d o x a n \ Asta Pharma AG, Frankfurt am Main, Ger-
many) were used in this study. The lyophilized formula-
tions contain mannitol as a bulking agent and the
dry-filled preparations contain sodium chloride as an
excipient to reach tonicity after dissolution. Sterile
Water for Injection was added to these preparations to
give an initial concentration of 20 mg/mL. The time to
obtain complete dissolution, under agitation, was deter-
mined visually by inspection of the solution for particles
against a white and dark background of a light box
equipped with an incandescent lamp and polarizing
filter. The solutions were either stored in glass vials,
were transferred to polyvinylchloride (pvc) syringes or
were diluted further with 0.9% sodium chloride or 5%
dextrose solutions to give an initial cyclophosphamide
concentration of 1 mg/mL. The test solutions were
stored at ambient room temperature (20-22°C), 4°C in
the refrigerator and at 37°C, respectively. The solutions
were either protected from light by wrapping in tinfoil or
they were exposed to normal room fluorescent light in a
day-night cycle. The day-night cycle consisted of 8 hours
in the dark and 16 hours in the light per day. The diluted
solutions (initial drug concentration: 1 mg/mL) were
Figure 1—Chemical structures of cyclophosphamide (I) and de-
stored in glass bottles or pvc minibags. Immediately gradation products (II—V). II: N-(2-hydroxyethyl)-N-
after sample preparation and at specific times after (3-hydroxypropyl)ethylenediamine; III: N-(2-hydroxyethyl)
-N'-(3-phosphatopropyi)ethylenediamine;
storage (1, 2. 3. 5 and 7 days) 20 |xL samples were taken IV: N-(3-hydroxypropyl)piperazine; V: N-(2-chioroethyl)
and analyzed for the content of undegraded cyclophos- -N'-(3-phosphatopropyl)ethylenediarnine(8).

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Local irritation of the veins can occur bv the use of a
co-solvent like ethanoi. The freeze-dried formulated
product has the advantage that it dissolves more rapidlv
after the addition of water. The time necessarv to
reconstitute the iyophilized product was 20 ± 5 seconds
(« = 12) while, the dissolution time for the dry-filled
formulation was 185 ± 23 seconds for EndoxanK-(« = 12)
and 210 ± 18 seconds for Cycloblastine* (n = 12).
The chemical stability of cyclophosphamide has been
the subject of many investigations (3-10). The degrada-
tion of the drug in aqueous solution can be described by
(pseudo) first order kinetics and in the pH region 2—10
the degradation rate is independent of pH and is mainly
subject to solvent catalysis. At pH values <2 and > 10
the degradation rate increases proportionally with [H*]
and [OH - ], respectively. Friedman and co-workers have
proposed the degradation mechanism (6) which was
confirmed later by Zon et al. (10), using high-resolution
nuclear magnetic resonance spectroscopy. The degrada-
tion starts with a rate-limiting intramolecular displace-
ment of a chloride ion whereby a carbon-chlorine bond
is cleaved followed by sequential P-N. C-Cl and P-0
bond hydrolysis to give various products (8. 10). The
chemical structures of these degradation products are
shown in Figure 1. The HPLC method used in this study
allows the selective determination of cyclophosphamide.
A relatively low analytical wavelength (214 nm) is
necessary as the compound has no L'v absorbing capaci-
ties in the higher regions. The assay is linear in the
concentration range of interest (0.5-20 mg/mL). The
equation of the calibration line isy = 24.26 (± 0.07)x +
6.6 (± 11.7) (A = 12) where y represents integrator peak
area units and x is the concentration of cyclophospha-
mide in mg/mL. Relative standard deviations of 1.3%
and 1.4% were obtained for replicate injections of the
lower and upper limits of the standard curve, respec-
tively. Figure 2 demonstrates typical chromatograms of a
blank (Figure 2A) and cyclophosphamide solutions at
; = 0 (Figure 2B) and / = 7 days at 4°C (Figure 2C) and
at 37°C (Figure 2D), recorded at a high sensitivity level,
to track down also minor impurities and degradation
products. Cyclophosphamide elutes in this system with a
retention time of approximate 6 minutes. The peaks in
the front (0.7 minutes) are also present in the blank and
originate from the solvent. The peak with retention time
of 1.21 minutes is already present in the pharmaceutical
product (Figure 2B); the identity of this product is not
known. At the stage of 50% degradation additional

Figure 2—HPLC chromatograms. recorded with a high sensitivity level, of a 20 p.L sample of water (blank) (A), cyclophosphamide
(20 mg/mL) in water at f = 0 (B), at í = 7 days stored at 4°C (C) and at f = 7 days stored at 37°C (D). The retention time of cyclophosphamide
is about 6 mins.

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TABLE 1
Stability of Cyclophosphamide in Sterile Water for Injection at Initial Concentration: 20 mg/mL
Percentage of Initial Concentration
Container Light at Indicated Time- (days)
FormulatiorT Material Conditions' Temp." 0 1 2 3 5 7
A elass dark 4=C 100 100.6 ± 1.9 100.6 * 1.7 99.6=: 1.7 99.6 ± 1.1 98.8 ± 10
B glass dark 4°C 100 99.9 ± 1.4 100.1 ± 1.8 99.9 ± 1.1 100.3 ± 1.2 99.8 ± 1.6
A PVC dark 4°C 100 100.4 ± 1.2 99.3 ± 1.1 98.9 ± 0.8 98.8 ± 0.8 99.0= 1.4
B PVC dark 4°C 100 100.5 ± 0.7 99.0 ± 0.8 98.9 * 1.4 99.9 ± 0.4 101.3 it 1.0
A glass dark ambient 100 100.2 ± 1.3 99.5 ± 1.4 96.8 ± 1.3 93.7 ± 2.0 92.7=: 1.9
B glass dark ambient 100 98.8 ± 0.5 97.2 ± 0.8 96.3 ± 0.2 92.8 ± 1.0 91.3 ± 0.3
A PVC dark ambient 100 97.5 * 1.6 96.3 * 1.3 96.6 ± 0.5 93.8 ± 1.3 91.3 ± 1.5
B PVC dark ambient 100 96.9 ± 0.5 96.5 ± 0.6 93.8 ± 0.5 90.3 ± 1.0 89.9 ± 0.8
A glass dark/light ambient 100 98.5 ± 1.5 98.1 ± 1.0 96.8 ± 1.2 95.2 ± 0.9 92.7 ± 2.0
B glass dark/light ambient 100 99.0 ± 0.9 97.6 ± 0.9 96.0 ± 0.9 93.4 ± 1.1 92.7 ± 0.8
A PVC dark/light ambient 100 99.3 ± 0.8 98.6 ± 1.7 95.9 ± 1.6 93.7 ± 0.6 92.1 ± 0.4
B PVC dark/light ambient 100 99.1 ± 1.9 97.0 ± 0.7 96.8 ± 1.4 93.1 ± 1.5 91.9 ± 1.0
A glass dark 37°C 100 90.7 ± 0.1 83.0 ± 1.2 74.2 ± 2.9 53.9 ± 2.5 46.7 ± 2.1
B glass dark 37°C 100 9 1 . 9 * 1.0 83.1 ± 2.1 74.4 i 1.0 53.3 ± 1.6 45.4 ± 1.3
A PVC dark 37°C 100 91.3 ± 1.1 85.5 ± 2.0 76.3 ± 0.4 53.3 ± 1.3 46.7 ± 0.9
B PVC dark 37°C 100 90.1 ± 0 . 7 81.1 ± 0.5 72.3 ± 0 . 1 49.4 ± 1.9 44.2 ± 1.0
" Results are reponed as mean ^: S.D. of three determinations.
h
Formulation A: drye-filled formulation (Endoxan--). Formulation B: lyophilized formulation (Endoxan*).
' Dark-light refers to normal roomfluorescentlight conditions in a day-night cycle.
J
Ambient temperature averaged between 20-22°C.

peaks, in particular a large peak in the solvent front,
appear in the chromatograms as can be seen in Figure
2D.
The results from the chemical stability testing of
cyclophosphamide after reconstitution are shown in
Table 1. It can be concluded that the nature of container
material (glass or PVC) has no influence on the chemical
stability of the drug under these conditions which is in
accordance with literature data (11). Normal room
fluorescent light also shows no effect on the overall
stability under these conditions. When 5e*- degradation
is taken as the limit for acceptability it is clear from
Table I that, after reconstitution, two days is a safe
margin to store cyclophosphamide solutions at room
temperature. Storage at 37°C results in a rapid degrada-
tion with a half-life of about five days and clearly
demonstrates the influence of higher temperatures on
the degradation rate. Cyclophosphamide solutions are.
therefore, not suitable for continuous infusion to ambu-
latory patients via pumping devices where the drug

TABLE 11
Stability of Cyclophosphamide in 0.9% Sodium Chloride at Initial Concentration: 1 mg/mL
Percentage of Initial Concentration
Container Light at Indicated Time" (days)
Formulation'' Material Conditions' Temp.-' 0 1 2 3; 5 7
A glass dark 4°C 100 101.2 ± 1.9 99.9 ± 1.4 99.5 ± 1.1 101.1 ± 0.4 102.0 ± 1.5
B glass dark 4°C 100 99.2 ± 1.4 97.3 ± 1.6 99.3 ± 1.7 97.9 ± 1.8 94.7 ± 1.7
A PVC dark 4°C 100 98.8 ±0.2 97.7 ± 0.8 98.1 ± 0 . 7 100.9 ± 0.5 99.6 ± 1.4
B PVC dark 4°C 100 101.9 ± 1.5 100.2 ± 1.1 99.2 ± 2 . 0 97.1 ± 2.1 97.1 ± 0.9
A glass dark ambient 100 100.0 ±0.6 97.8 ± 1.9 96.7 ± 1.5 97.8 ± 1.4 96.3 ± 1.9
B glass dark ambient 100 99.4 ±0.9 98.7 ± 0.6 98.7 ± 1.0 99.5 ± 1.3 97.8 ± 1.9
A PVC dark ambient 100 99.6 ± 0 . 8 99.7 ± 0.9 95.5 ± 0 . 7 96.7 ± 2.1 96.4 ± 0.9
B PVC dark ambient 100 100.7 ±0.8 99.5 ± 0.3 98.1 ± 2 . 8 97.6 ± 0.3 96.5 ± 0.4
A glass dark/light ambient 100 99.9 ± 1.8 97.2 ± 0.5 92.8 ± 2 . 4 86.7 ± 0.7 86.9 ± 2.1
B glass dark/light ambient 100 101.7 ± 1.2 97.7 ± 1.5 95.1 ± 1.2 90.8 ± 0.2 90.1 ± 0.3
A PVC dark/light ambient 100 100.9 ± 1.9 98.6 ± 1.3 101.1 ± 1.1 96.8 ± 2.3 96.7 ± 1.9
B PVC dark/light ambient 100 102.0 ±2.1 100.9 ± 1.4 100.0 ± 1.7 98.0 ± 0.8 97.5 ± 1.7
A glass drk 37°C 100 94.0 ± 0 . 8 88.9 ± 2.0 76.9 ± 0 . 8 55.6 ± 2.3 50.1 ± 1.6
B glass dark 37°C 100 98.7 ± 1.3 86.7 ± 1.7 79.1 ±3.1 60.1 ± 1.6 49.3 ± 1.1
A PVC dark 37°C 100 93.0 ± 0 . 7 77.7 ± 0.8 69.9 ± 2 . 2 56.7 ± 2.3 49.9 ± 0.9
B PVC dark 37°C 100 91.6 ±0.5 85.2 ± 0.2 68.0 ± 1.1 57.0 ± 0.9 51.5 ± 0.9
" Results are reported as mean ± S.D. of three determinations.
* Formulation A: drye-filled formulation (Endoxan*). Formulation B: lyophilized formulation (Endoxan*).
r
Dark/light refers to normal roomfluorescentlight conditions in a day-night cycle.
a
Ambient temperature average between 20-22°C.

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TABLE III
Stability of Cyclophosphamide in 5% Dextrose at Initiai Concentration: 1 mg/mL
Percentage of 1Initial Concentration
Container Light at Indicated Time : (days)
Formulation* Material Conditions' Temp. J 0 1 2 3 5 7
A glass dark 4°C 100 98.8 * 0.8 99.9 ± 1.2 98.7 * 1.2 102.9 ¿ 1.5 101.3 ± 1.2
B glass dark 4°C 100 96.3 ± 2.0 97.0 ± 1.9 98.8 ± 0.8 97.8 ± 1.9 95.4 ± 1.9
A PVC dark 4°C 100100.8 ± 1.6 98.9 ± 1.4 98.8 ± 1.5 99.6 í 1.1 100.1 i 1.4
B PVC dark 4°C 100 99.2 * 0.7 98.4 ± 1.3 98.2 ± 0.9 98.4 ± 1.2 97.4 ± 0.4
A glass dark ambient 100100.5 * 1.0 98.6 ± 1.3 98.1 ± 1.2 99.1 i 1.4 97.2 * 1.2
B glass dark ambient 100 98.4 ± 0.8 98.2 ± 2.1 97.8 ± 1.3 99.2 ± 1.1 95.8 * 0.6
A PVC dark ambient 100 98.3 + 1.2 96.3 ± 0.9 92.0 = 0.7 91.0 ± 0.9 90.5 * i.O
B PVC dark ambient 100 97.4 ± 2.3 96.9 ± 0.9 95.3 ± 1.8 93.0 ± 1.7 92.8 ± 0.9
A glass dark/light ambient 100 99.9 i 0.9 100.0 ± 1.2 98.5 ± 1.9 95.4 ± 0.3 94.9 ± 2.3
B glass dark/light ambient 100 97.9 ± 2.3 95.8 ± 1.3 96.4 £ 1.9 94.9 i 0.7 95.0 ± 2.4
A PVC dark/light ambient 100 99.4 ± 0.2 97.5 ± 1.0 97.2 * 1.2 95.8 i 0.9 95.5 ± 1.7
B PVC dark /light ambient 100 99.3 ± 1.7 97.4 * 2.1 94.3 ± 0.7 94.0 ± 2.5 93.4 ± 1.9
A giass dark 37°C 100 90.5 ± 1.7 85.0 ± 0.4 79.8 ± 1.9 56.8 i 1.8 51.0 ± 1.4
B glass dark 37°C 100 83.1 ± 1.8 75.9 ± 3.5 65.0 ± 2.2 51.3 ± 1.3 44.7 ± 1.3
A PVC dark 37°C 100 92.2 ± 0.9 84.9 ± 1.7 76.3 ± 0.7 57.6 ± 1.0 50.1 ± 1.0
B PVC dark 37°C 100 95.5 ± 0.4 84.0 ± 2.2 77.3 ± 2.4 54.3 ± 1.3 49.2 ± 2.4
" Results are reported as mean ± S.D. of three determinations.
" Formulation A: drye-filled formulation (Endoxan^). Formulation B: lyophilized formulation (Endoxan").
' Dark/light refers to normal room fluorescent light conditions in a day-night cycle.
"' Ambient temperature averaged between 20-22°C.

solution stays for some days at body temperature. In a
concentration of 1 mg/mL. cyclophosphamide is stable
for seven days at 4°C in admixtures with 0.9% sodium
chloride or 5% dextrose (Tables II and III); taking into
account the standard deviation of the test results, the
reconstituted lyophilized formulation (in glass and in
the dark), where we found 94.7 ± 1.7% of the initial
concentration after seven days, was also defined as
stable. From the experiments at ambient temperature it
was found that cyclophosphamide is stable ( < 5% degra-
dation) in 0.9% sodium chloride when stored in the dark
in both glass as well as pvc (Table II). However, under
normal room fluorescent light conditions and further
similar conditions (ambient temperature: drug concen-
tration 1 mg/mL: in 0.9% sodium chloride) the drug is
not stable when stored in glass ( > 5% degradation after
seven days) but it is stable in PVC. This observation may
indicate that cyclophosphamide is susceptible to light-
catalyzed degradation under these conditions while pvc
offers some protection. More research is necessary to
obtain a clear insight into this phenomenon. Other
factors are probably involved since the influence of the
container material for the 5% dextrose solution was
observed under storage in the dark at ambient tempera-
ture (PVC: >5% degradation after seven days; glass:
<5% degradation after seven days). There were no
significant differences between the data obtained in 5%
dextrose in PVC or glass under normal room fluorescent
light conditions.
The solutions were also visually inspected for clarity,
color and signs of precipitation using the light box.
There were no changes of these parameters from t = 0
and after the storage period of seven days under the
conditions tested.
In conclusion the results of these stability studies
demonstrate that:
— the freeze-dried formulation of cyclophosphamide
dissolves more rapidly than the dry-filled products:
— there are no differences between the stability of
cyclophosphamide solutions obtained from both types
of formulations;
— cyclophosphamide solutions are stable ( <5% degra-
dation) when stored in glass or PVC in concentrations
1 or 20 mg/mL at 4°C. At 37°C rapid degradation
occurs (the half-life is about seven days);
— after dilution of the reconstituted solution with 5%
dextrose or 0.9% sodium chloride cyclophosphamide
(concentration 1 mg/mL) is stable for at least two
days at ambient temperature in both glass and PVC
and under normal room fluorescent light conditions
in a day-night cycle or in the dark.
Acknowledgements
This study was supported by a grant from the Nijbak-
ker Morra Foundation, Amsterdam, The Netherlands.
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Journal of Parenteral Science & Technology
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