Full text of DOI:10.1093/chromsci/41.2.57

Journal of Chromatographic Science, Vol. 41, February 2003
Rapid Analysis of Phentolamine by High-Performance
Liquid Chromatography
Gregory K. Webster*, Robert R. Lemmer, and Steven Greenwald
Summit Analytical Services, 1467 Summerfield Lane, Howell, MI 48843
methanol (at an apparent pH of 4.0)/48% of 6.16mM octanesul-
fonic acid (with 1% acetic acid aqueous phase at pH 4.0). Using a
Abstract
UV detection wavelength of 280 nm and antazoline and naphazo-
line as internal standards, the de Bros and Wolshin system estab-
lished an average recovery of 85% over a 15–5000 ng/mL
phentolamine range. The Godbillon and Carnis system used a
Lichrosorb R-18 or R-8 (10 µm) column for urine samples and a
Lichrosorb R-8 (5-µm) column for plasma samples. The mobile
phase for this system was 60% 2.6mM orthophosphoric acid–40%
acetonitrile (flowing at 3 mL/min for urine samples) and 85%
HCl-sodium citrate buffer (at pH 4.0)–15% acetonitrile flowing at
2 mL/min for plasma samples. The Godbillon and Carnis system
posted a sensitivity limit of 200 ng/mL phentolamine for plasma
samples and 2–3 µg/mL phentolamine for urine samples using
UV detection at 254 nm.
A rapid liquid
chromatographic method is validated for the quantitative analysis of
phentolamine. Phentolamine exists in three forms for this
investigation: as a mesylate salt, hydrochloride salt, and free base. In
solution, phentolamine dissociates from its salt and is
chromatographed as free phentolamine. This validation confirms the
analysis of each form, which is simply based upon molar mass
differences encountered in weighing. As such, both the United
States Pharmacopeia hydrochloride and mesylate standards are used
throughout this validation to demonstrate this equivalency. The
validation demonstrates that this method may be used to quantitate
phentolamine, regardless of its salt form.
Phentolamine was determined in serum and liver homogenate
samples by Kerger et al. (6). The system used a Phase-2 ODS
column and a mobile phase of 75% of a 0.15M monochlororac-
etate solution (at pH 3.0)–25% acetonitrile spiked with 0.35 g/L
EDTA. The mobile phase flowed at 0.6 mL/min and electrochem-
ical detection was carried out at +900 mV. The Kerger et al.
system yielded a detection limit of 5 ng/mL phentolamine in
serum and 10 ng/mL in liver.
Introduction
Phentolamine mesylate is an antihypertensive agent sold by
Ciba Geneva (Summit, NJ) under the registered trade name of
Regitine. The drug produces an alpha-adrenergic block of rela-
tively short duration. Phentolamine mesylate also has vasodilator
effects on vascular smooth muscle.
The chemical stability of phentolamine mesylate and
papaverine HCl formulation solutions was studied by LC by
Torrado-Valeiras et al. for i.v. injections. The system used a C18
column, UV detection at 254 nm, and a mobile phase consisting
of 65% methanol–35% of a 1% acetic acid solution adjusted to pH
4.0 with triethylamine. Torrado-Valeiras et al. found that the
chemical degradation of phentolamine and papaverine dissolved
in 5% glucose and stored for 2 months at 5, 30, 45 and 60ºC, and
after 4 years at room temperature it was less than 10%.
LC has proven to be an effective tool for the quantitative anal-
ysis of phentolamine. However, compendial potency determina-
tions are still based on titrimetic determinations (12). Because LC
is much more selective, stability-indicating, and can be used to
analyze for impurities, a rapid LC analysis for phentolamine was
developed to replace the compendial titrimetic assay.
Phentolamine was found to be effectively resolved from its syn-
thesis precursors using an ODS-AQ column (Waters, Milford, MA)
and an isocratic mobile phase consisting of a heptanesulfonic acid
Chromatographic analysis of phentolamine has consisted of gas
(1), thin-layer (2), and liquid chromatographic (LC) (3–11) deter-
minations. Gas chromatographic (GC) analyses require a deriva-
tization step in order to increase the volatility of phentolamine. As
such, LC has a distinct advantage over GC in terms of sample
preparation.
Mollica et al. (3) developed an LC method scheme for the anal-
ysis of imidazoline drugs. For phentolamine analysis, the scheme
used a strong cation exchange column and a pH 11.45 buffer at
0.8 mL/min and UV detection at 254 nm. The method was not
investigated for the resolution of the synthesis precursors of
phentolamine.
de Bros and Wolshin (4) and Godbillon and Carnis (5) used LC
in blood and urine determinations. The de Bros and Wolshin
system used a µBondapak C18 column and an ion pairing mobile
phase flowing at 2.4 mL/min. The mobile phase was 52%
* Author to whom correspondence should be addressed: email SummitAnalytical@netscape.net.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
57

Journal of Chromatographic Science, Vol. 41, February 2003
buffer, methanol and acetonitrile in a ratio of 35:55:10 (v/v/v).
Phentolamine exists in three forms for this investigation: (a) as
a mesylate salt, (b) a hydrochloride salt, and (c) a free base. In
solution, phentolamine dissociates from its salt and is chro-
matographed as free phentolamine. The analysis of each form is
simply based upon molar mass differences encountered in
weighing. Unlike the United States Pharmacopeia (USP) titration
assay, LC presents the opportunity to quantitate all three salt
forms. The ability to assay phentolamine regardless of its salt
form is important for reaction monitoring and quality control. As
such, the use of both the USP hydrochloride and mesylate stan-
dards are used throughout this validation to demonstrate this
equivalency.
a 2-L flask, which contained approximately 1 L of H20. Upon total
dissolution, the solution was adjusted to a pH of 3.0 using 2N
H2SO4 and diluted to mark with H2O. For the chromatographic
mobile phase, the buffer was diluted with methanol and acetoni-
trile in a ratio of 35:55:10 (v/v/v), respectively.
Sample and standard preparation
The samples and standards were prepared to a concentration of
0.1 mg/mL in a dilution solution (15mM heptanesulfonic acid
buffer and 500 mL methanol in a ratio of 1:1, v/v).
Validation materials
The standards were the current USP standards for phento-
lamine hydrochloride (Lot F) and phentolamine mesylate
(Lot H). The phentolamine free base; phentolamine hydrochlo-
ride; phentolamine mesylate; 2-chloromethylimidazoline
HCl,4,5-dihydro-2-[N-(m-hydroxyphenyl)-N-phenylamino-
methyl]-1H-imidazole; 3-hydroxy-4'-methyldiphenylamine;
3-acetoxy-4'-methyldiphenylamine; and 3-acetoxy-4'-methyl-
diphenylaminoacetonitrile samples used for the method valida-
tions were all synthesized for this study. The structures for these
compounds are given in Figure 1.
Experimental
LC
The LC data reported was generated using three equivalent sys-
tems: (a) HP Series 1050 system with an HP Series 1050 PDA
photodiode array detector (Hewlett-Packard, Palo Alto, CA), (b)
HP Series 1050 system equipped with an HP Series 1050 UV
detector, and (c) Alliance 2690 Module equipped with a Waters
486 UV detector. All three systems ran at ambient temperature
and with an injection volume of 10 µL. An ODS-AQ, 5-µm, 120 A,
4.6 × 150 mm C18 column phase I and a Metasil AQ 5-µm, 120 A,
4.6 × 150 mm C18 column phase II (Metachem, Torrance, CA)
were used interchangeably in this study. All columns were used
with a flow rate of 1.0 mL/min and with the detector wavelength
set at 220 nm.
Results and Discussion
Phentolamine synthesis
The synthesis of 4,5-dihydro-2-[N-(m-hydroxyphenyl)-N-
(p-methylphenyl)amino-methyl]-1H-imidazole (phentolamine)
was reported in 1950 by Urech et al. and patented in the same
year by Miescher (15). The patent disclosed two methods of
preparing phentolamine hydrochloride. The first example
described the reaction of 3-hydroxy-4'-methyldiphenylamine
with 2-chloromethylimidazoline hydrochloride to yield phento-
Mobile Phase
The mobile phase buffer (15mM heptanesulfonic acid) con-
sisted of 6.06 g heptanesulfonic acid and sodium salt weighed into
4,5-dihydro-2-[N-(m-hydroxphenyl)-N-phenylamino-methyl]-1H-imidazole
Figure 1. Structures for compounds of interest including phentolamine (A), 2-chloromethylimidazoline HCl (B), 3-hydroxy-4'-methyldiphenylamine (C), 4,5-dihydro-
2-[N-(m-hydroxyphenyl)-N-phenylamino-methyl]-1H-imidazole (D), 3-acetoxy-4'-methyldiphenylamine (E), and 3-acetoxy-4'-methyldiphenylaminoacetonitrile.
58

Journal of Chromatographic Science, Vol. 41, February 2003
lamine hydrochloride. Specifically, Miescher described a process
in which two equivalents of 3-hydroxy-4'-methyldiphenylamine
and one equivalent of chloromethylimidazoline hydrochloride
were heated at 150ºC for 16 h under a nitrogen atmosphere. The
viscous liquid was then cooled to less than 100ºC and partitioned
between water and ethyl acetate. The desired product was
obtained from the aqueous layer upon cooling. No yields were
cited in the patent.
In U.S. Patent 2,503,059, Miescher described an alternate
method of synthesizing phentolamine from 3-hydroxy-4'-
methyldiphenylamine. This procedure built the imidazoline ring
onto the diphenylamine in three steps. First, the sodium salt of
3-hydroxy-4'-methyl-diphenylamine was acetylated with acetyl
chloride to yield 3-acetoxy-4'-methyldiphenylamine. The
3-acetoxy-4'-methyldiphenylamine was then reacted with
paraformaldehyde and potassium cyanide in aqueous acetic acid
to give 3-acetoxy-4'-methyldiphenylaminoacetonitrile. Finally,
the nitrile was heated with ethylenediamine in the presence of a
catalytic amount of hydrogen sulfide (or carbon disulfide) to pro-
duce phentolamine. The reaction mixture was partitioned
between ethyl acetate and dilute hydrochloric acid. Phentolamine
hydrochloride was obtained after concentration of the aqueous
layer.
The inventors published a variation of the first method (14)
Figure 3. LC analysis of phentolamine in the presence of potential synthesis
materials using column phase II. The peaks represent 2-chloromethylimida-
zoline HCl (A), 4,5-dihydro-2-[N-(m-hydroxyphenyl)-N-phenylamino-
methyl]-1H-imidazole (B), phentolamine (C), 3-hydroxy-4'-methyl-
diphenylamine (D), 3-acetoxy-4'-methyldiphenylamine (E), and 3-acetoxy-
4'-methyldiphenylaminoacetonitrile (F).
Figure 2. LC analysis of phentolamine in the presence of potential synthesis
materials using column phase I. The peaks represent 2-chloromethylimidazo-
line HCl (A), 4,5-dihydro-2-[N-(m-hydroxyphenyl)-N-phenylamino-methyl]-
1H-imidazole (B), phentolamine (C), 3-hydroxy-4'-methyldiphenylamine (D),
3-acetoxy-4'-methyldiphenylamine (E), and 3-acetoxy-4'-methyldipheny-
laminoacetonitrile (F).
Table I. LC Accuracy Runs of Phentolamine Salts vs. USP Assay
LC system 1
column phase I
LC system 2
column phase I
LC system 2
column phase I
LC system 3
column phase II
USP titration
assay (%)
Phentolamine salt
HCl standard
Mesyl standard
HCl standard Mesyl standard
HCl standard
Mesyl standard
HCl standard
Mesyl-1
Mesyl-2
Mesyl-3
Mesyl-4
Base-5
Base-6
Base-7
Base-8
HCl-9
100.5
99.1
99.4
99.8
100.1
99.2
100.0
99.5
96.5
90.8
99.3
94.8
99.9
99.1
99.4
99.3
98.8
97.9
98.7
98.2
95.0
89.4
97.8
93.3
98.7
98.5
98.9
97.9
98.4
97.9
96.7
97.8
94.9
89.6
97.2
92.9
99.2
99.0
99.4
98.4
98.4
97.9
96.7
97.8
95.3
89.9
97.5
93.1
100.5
98.6
99.2
98.5
99.2
97.8
97.5
97.8
93.6
89.8
97.0
95.2
98.3
97.7
98.3
97.6
98.3
97.0
96.7
97.0
92.9
89.1
96.3
94.5
98.7
98.7
98.8
98.4
98.5
98.1
97.7
98.0
95.6
90.1
97.4
93.9
99.0
100.5
100.7
97.7
HCl-10
HCl-11
HCl-12
59

Journal of Chromatographic Science, Vol. 41, February 2003
whereby phentolamine hydrochloride was obtained by reacting
equimolar amounts of chloromethylimidazoline hydrochloride
and 3-hydroxy-4'-methyldiphenylamine in o-dichlorobenzene.
After heating at reflux for 6 h, the gummy mass was filtered and
recrystallized from water to produce phentolamine hydrochloride
(no yield cited). The authors stated that xylenes were an accept-
able substitute for dichlorobenzene. Phentolamine mesylate was
obtained from the phentolamine hydrochloride by a simple salt
switch.
pounds were greatly improved. The mobile phase conditions were
optimized to be heptanesulfonic acid buffer, methanol, and ace-
tonitrile in a ratio of 35:55:10 (v/v/v). LC columns from several
column manufacturers were tested for performance. The ODS-
AQ and an equivalent phase from Metachem were chosen because
they produced the best resolution and peak shape performance
(Figures 2 and 3).
Linearity of response/detection limits
The system linearity for phentolamine was validated to span
approximately 50–150% of the nominal sample preparation used
in the standard preparation. Each LC system produced a correla-
tion coefficient (r) greater than 0.99. The worst limit of detection
(LOD) (S/N = 3) and limit of quantitation (LOQ) (S/N = 10) were
obtained for the photodiode array system. This system had an
LOD of 1.75 ng/mL and an LOQ of 5.83 ng/mL of phentolamine
using the USP mesylate standard.
LC analysis/validation
The methods established in the literature (3–7), as well as in-
house data from similar studies, were used as a starting point for
the analytical development work. The previous methods required
higher mobile phase flow rates to yield short analysis times, and
it was not established that the LC method would resolve phento-
lamine from its synthesis precursors. The latter requirement is
necessary for a practical quality control procedure.
The de Bros and Wolshin (4) system established that an ion-
pairing mobile phase was effective in producing a good peak
shape with phentolamine. After optimizing the LC system with
heptanesulfonic acid buffer–methanol gradient investigations, as
suggested by Snyder et al. (13), the optimal resolution and peak
shape of phentolamine was still unsuitable. Acetonitrile was
added to the mobile phase and the peak shapes of the test com-
Accuracy
The accuracy of the LC method was investigated by comparing
the analysis results of the USP titration assay for phentolamine
mesylate with the chromatographic result (Table I). Because the
USP titration procedure does not work for the hydrochloride or
free base salts because of solubility differences in the specified
USP assay solvents, the LC and titration results could not be
Table II. Statistical Comparison of LC System Results vs.
USP Assay for Phentolamine Mesylate*
Table IV. Method Precision
USP
mesylate
standard
LC system
USP
Column
texp
USP HCl
standard
LC system
Phentolamine salt
1
2
3
Phentolamine HCl
Phentolamine mesylate
Phentolamine HCl
Phentolamine mesylate
Phentolamine HCl
Phentolamine mesylate
Phentolamine HCl
Phase i
Phase i
Phase i
Phase i
Phase i
Phase i
Phase ii
0.24
0.08
1.79
0.88
0.34
2.30
1.32
1
HCl-9
Average
95.41
0.14
0.15
98.21
0.2
95.52
0.18
0.19
98.81
0.24
0.25
99.18
0.18
0.18
96.04
0.34
0.35
99.48
0.14
0.14
98.93
0.14
0.14
94.64
0.47
0.5
Standard deviation
%RSD
Base-5
Mesyl 13
HCl-9
Average
Standard deviation
%RSD
Average
Standard deviation
%RSD
4
0.21
98.62
0.18
0.19
95.73
0.21
0.22
98.88
0.13
0.13
98.42
0.13
0.13
95.52
0.52
0.55
98.8
0.55
0.56
100.21
0.21
0.21
* tcrit = 3.18.
2
Average
Table III. Standard Addition Investigations
Standard deviation
%RSD
Phentolamine
salt
Sample
recovery (%)
Spike
recovery (%)
Base-5
Average
LC (%)
Standard deviation
%RSD
Mesyl-13 Average
Standard deviation
Mesyl-2
Mesyl-2 spike
Mesyl-1
Mesyl-1 spike
HCl-12
HCl-12 spike
HCl-11
HCl-11 spike
Base-7
Base-7 spike
Base-5
101.9
121.4
100.4
119.3
93.8
108.8
97.6
113.3
97.8
101.3
98.8
93.6
97.5
95.9
96.9
99.4
98.4
99.8
99.9
98
%RSD
3
HCl-9
Average
Standard deviation
%RSD
Base-5
Mesyl 13
Average
Standard deviation
%RSD
97.67
0.56
0.57
98.83
0.24
0.24
112.1
99.3
113.7
Average
Standard deviation
%RSD
Base-5 spike
99.4
60

Journal of Chromatographic Science, Vol. 41, February 2003
directly compared for these salts. The accuracy investigations
were conducted using both the USP mesylate and hydrochloride
standards for comparison.
No significant difference in results was detected for the LC anal-
ysis of phentolamine mesylate and the USP titration assay. The
analysis results for the phentolamine mesylate accuracy samples
run on the three LC systems were investigated using a two-tailed
paired t-test and a 95% confidence level (Table II). Each system
and the corresponding USP standard used were compared with
the compendial USP assay (titration) result. For the statistical
comparison, the critical t-value was 3.1824. Thus, sample sets
yielding a paired t-value less than this critical value were deemed
“not significantly different.” The data illustrate that no significant
difference was detected using either the USP standard on the
chromatographic systems or the USP compendial assay result. No
significant difference was detected between the use of either the
ODS AQ (Table II, phase I) or Metasil AQ (Table II, phase II)
columns versus the compendial USP assay (titration) result.
Standard addition study
Because the USP titration cannot access the potency of the
hydrochloride salt or base, the accuracy of the LC method was
further investigated for all three salt forms by spiking representa-
tive samples with phentolamine mesylate and establishing the
recovery. All recoveries, regardless of salt complex, were 98% or
better (Table III). Because this investigation illustrates equiva-
lency in the LC assay of phentolamine regardless of its salt (mesy-
late, free base, or hydrochloride), this method, unlike the USP
assay, can be used to quantitate any of these salts whereas the USP
titration can be used solely for the mesylate salt.
Table V. System Precision
USP
mesylate
standard
USP HCl
standard
LC system
Phentolamine salt
1
HCl-9
Average
95.3
0.1
0.1
98.5
0.7
0.7
98.0
0.1
0.1
95.8
0.1
0.1
99.3
0.1
0.1
98.4
0.2
0.2
95.4
0.1
0.08
99.7
0.3
0.3
100.8
0.8
95.7
0.1
0.1
99.1
0.7
0.67
98.8
0.1
0.1
96.2
0.1
0.1
99.7
0.1
0.1
98.9
0.2
0.2
94.3
0.1
0.1
98.5
0.3
0.3
99.6
0.8
Standard deviation
%RSD
Base-5
Mesyl 13
HCl-9
Average
Standard deviation
%RSD
Average
Standard deviation
%RSD
Precision
Method precision was determined by analyzing five weights of a
single phentolamine mesylate, free base, and hydrochloride lot
for each LC system (Table IV). System precision for each LC
system was obtained by analyzing six consecutive injections of a
single phentolamine mesylate, free base, and hydrochloride
sample preparation (Table V). All three LC systems exhibited
acceptable precision per USP.
2
Average
Standard deviation
%RSD
Base-5
Mesyl-13
HCl-9
Average
Standard deviation
%RSD
Average
Standard deviation
%RSD
Ruggedness
The ruggedness of the LC method was determined by analyzing
accuracy (Table I, system 2), method precision (Table IV, system
1), and system precision (Table V, system 1) using separate LC sys-
tems, ODS-AQ column, and an analyst. For this section, analyst 2
ran linearity and accuracy on LC system 2 and precision on LC
system 1. Method precision for ruggedness was again determined
by analyzing five weights of a single phentolamine mesylate, free
base, and hydrochloride lot for each LC system. The LC method
exhibited acceptable precision per USP.
3
Average
Standard deviation
%RSD
Base-5
Mesyl 13
Average
Standard deviation
%RSD
Average
Standard deviation
%RSD
System suitability parameters
The chromatographic system was also tested to verify that the
0.8
0.8
Table VI. System Suitability Figures for Phentolamine and its Synthesis Materials
Resolution
Tailing
Theoretical plates
Compound
LC phase
I*
II†
I*
II†
I*
II†
2-Chloromethyllmidazline HCl
4,5-dihydro-2-[N-(m-hydroxyphenyl)-N-phenylamino-methyl]-1H-imidazole
Phentolamine
3-Acetoxy-4'-methyl diphenylaminoacetonitrile
3-Acetoxy-4'-methyl diphenylamine
3-Hydroxy-4'-methyl diphenylamine impurity
–
–
1.5
1.3
1.3
1.2
1.2
1.1
1.0
1.5
1.3
1.2
1.2
1.0
890
1144
1539
3102
4730
6080
544
892
1214
2892
3928
5897
2.9
1.9
4.2
5.0
4.8
2.3
1.6
4.8
3.1
5.8
* ODS-AQ column
† Metasil AQ column
61

Journal of Chromatographic Science, Vol. 41, February 2003
phentolamine peak maintained its integrity in the presence of the
potential starting materials used for its synthesis: 2-
chloromethylimidazoline; 4,5-dihydro-2-[N-(m-hydroxyphenyl)-
N-phenylamino-methyl]-1H-imidazole (an R&D synthesis
marker); 3-hydroxy-4'-methyldiphenylamine, 3-acetoxy-4'-
methyldiphenylamine; and 3-acetoxy-4'-methyldiphenylamino-
acetonitrile. The system suitability parameters for these
compounds using column phase i are presented in Table VI.
References
1. A. Sioufi, F. Pommier, P. Mangoni, S. Gauron, and J.P. Metayer. Gas
chromatographic determination of phentolamine in human plasma
and urine. J. Chromatogr., Biomed. Appl. 222 (3): 429–35 (1981).
2. I. Grabowska, D. Rajzer, A. Kraczkowska, and B. Kremska.
Qualitative and quantitative assessment of solution stability of phen-
tolamine hydrochloride. Acta Pol. Pharm. 47(3-4): 29–35 (1990).
3. J.A. Mollica, G.R. Padmanabhan, and R. Strusz. Liquid chromato-
graphic analysis of imidazolines. Anal. Chem. 45(11): 1859–62
(1973).
4. F. de Bros and E.M. Wolshin. Determination of phentolamine in
blood and urine by high performance liquid chromatography. Anal.
Chem. 50(3): 521–25 (1978).
5. J. Godbillon and G. Carnis. Determination of the major metabolite of
phentolamine in human plasma and urine by high performance
liquid chromatography. J. Chromatogr., Biomed. Appl. 222(3):
461–66 (1981).
6. B.D. Kerger, R.C. James, and S.M. Roberts. An assay for phento-
lamine using high performance liquid chromatography with electro-
chemical detection. Anal. Biochem. 170(1): 145–51 (1988).
7. J.J. Torrado-Valeiras, J. Montaner, and J.J. Torrado-Duran. Chemical
stability of phentolamine mesylate and papaverine. An. Real. Acad.
Farm. 1987(4): 547–551 (1991).
8. K.U. Ushbaev, A.U. Tulegenova, S.K. Zheterova, and Ya.R.
Rakhmatulin. Quantitative determination of phentolamine in tablets
using kinetic method. Farmatsiya (Moscow) 36(3): 40–42 (1987).
9. E.I. Egorova, E.A. Krasnov, and S.V. Terent’eva. Microcrystalloscopic
reaction identification of phentolamine ingredients in dyes.
Farmatsiya (Moscow) 34(3): 76–78 (1985).
Specificity study
A forced degradation study was performed to verify the separa-
tion of phentolamine and its potential degradation products. To
perform this study, a stock solution of phentolamine mesylate
[0.25283 g of reference material in 25 mL of 50% heptanesulfonic
acid (HSA)–50% methanol dilution solution] was diluted 1:10 in
1N HCl, 15mM heptanesulfonic acid, 1.25N NaOH, and 30% H2O2
as stress solvents. After an 18-h storage period at 60ºC, the sam-
ples were again diluted 1:10 in dilution solution and analyzed
neat by LC. The degradation samples were compared with a
freshly prepared standard preparation, and the percentage of
phentolamine remaining was determined. The peak purity factor
was confirmed using a Hewlett-Packard Series 1050 photodiode
array detector. Chromatographic integrity was maintained for
each stressed system. It was noted that the degradation peak pro-
duced with the HCl matrix eluted near the phentolamine peak;
the system retained a resolution of 0.9 between these peaks.
Under the degradation conditions used, the peak response for
phentolamine remained acceptably homogeneous.
10. Z. He and Y. Zhong. Quantification of phentolamine injections by
UV absorption with acidic colorant. Yaowu Fenxi Zazhi 12(6):
352–54 (1992).
11. W.E. Seifert, Jr., A. Ballatore, and R.M. Caprioli. Direct analysis of
drugs by continuous-flow fast-atom bombardment and tandem mass
spectrometry. Rapid Commun. Mass Spectrom. 3(4): 117–122
(1989).
12. USP XXIII, United States Pharmacopeial Convention, Rockville, MD,
1995.
13. L.R. Snyder, J.L. Glajch, and J.J. Kirkland. Practical HPLC Method
Development. Wiley & Sons, New York, NY, 1988.
14. E. Urech, A. Marxer, and K. Miescher. 2-Aminoalkyl-imdazoline.
Helv. Chim. Acta. 33: 1386–1407 (1950).
15. K. Miescher, assigned to Ciba. U.S. Patent 2,503,059, April 4, 1950.
Conclusion
The LC method presented here has been qualified and found
acceptable for use in the quantitative analysis of phentolamine as
its free base, hydrochloride, and mesylate salts. This method was
found to yield assay results not significantly different from the
USP compendial titration assay for phentolamine mesylate.
Whereas the USP procedure was ineffective at assaying the free
base and hydrochloride salt of phentolamine, the LC procedure
effectively validated and resolved phentolamine from its potential
starting materials and degradants.
Manuscript accepted November 27, 2002.
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