Chromatographia (2018) 81:1147–1162

https://doi.org/10.1007/s10337-018-3555-8

ORIGINAL

Kinetics and Characterization of Degradation Products

of Dihydralazine and Hydrochlorothiazide in Binary Mixture by HPLC-
UV, LC-DAD and LC–MS Methods
Anna Gumieniczek1 · Justyna Galeza1 · Tomasz Mroczek2 · Krzysztof Wojtanowski2 · Katarzyna Lipska1 ·
Rafał Pietras1

Received: 30 March 2018 / Revised: 7 June 2018 / Accepted: 11 June 2018 / Published online: 25 June 2018
© The Author(s) 2018

Abstract
Dihydralazine and hydrochlorothiazide were stored at high temperature and humidity, under UV/Vis light and different pH,
as individual drugs and the mixture. Then, a sensitive and selective HPLC-UV method was developed for simultaneous
determination of dihydralazine and hydrochlorothiazide in presence of their degradation products. Finally, the degradation
products were characterized through LC-DAD and LC–MS methods. Dihydralazine was sensitive to high temperature and
humidity, UV/Vis light and pH ≥ 7. At the same time, it was resistant to acidic conditions. Hydrochlorothiazide was sensitive
to high temperature and humidity, UV/Vis light and changes in pH. Its highest level of degradation was observed in 1 M HCl.
Degradation of the drugs was higher when they were stressed in the mixture. In the case of dihydralazine, the percentage
degradation was 5–15 times higher. What is more, dihydralazine became sensitive to acidic conditions. Hydrochlorothiazide
was shown to be more sensitive to UV/Vis light and pH > 4. Degradation of dihydralazine and hydrochlorothiazide followed
first-order kinetics. The quickest degradation of dihydralazine was found to be in 1 M NaOH while of hydrochlorothiazide
was in 1 M HCl (individual hydrochlorothiazide) or at pH 7–10 (hydrochlorothiazide in the mixture). A number of new
degradation products were detected and some of them were identified by our LC-DAD and LC–MS methods. In the stressed
individual samples, (phenylmethyl)hydrazine and 1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide were observed for the
first time. Interactions between dihydralazine and hydrochlorothiazide in the mixture were confirmed by additional degrada-
tion products, e.g., 2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1,4-trioxide.

Keywords Dihydralazine and hydrochlorothiazide · Kinetics of degradation · Degradation products · Interactions ·

HPLC-UV, LC-DAD and LC–MS methods

Introduction reduce the resistance in arterial vessels. Because of this

unique mechanism of action, they can be combined with
Hydralazine and its derivative dihydralazine are used in the other drugs of complementary effects, e.g., with hydrochlo-
treatment of hypertension in Europe, the USA and China rothiazide from diuretics. Two-component formulations of
[1]. Because of their special action in some types of hyper- hydralazine and hydrochlorothiazide are already present in
tension, they are included in European Pharmacopoeia the USA and China.
(Eur. Ph.) [2]. They both are arterial vasodilators that can Although two-component tablets present many advan-
tages, they introduce the problem of interactions between
the active ingredients. For example, degradation of one drug
* Anna Gumieniczek may be accelerated by another one. Interactions can also
anna.gumieniczek@umlub.pl lead to generate new degradation products. Therefore, the
1 main goal of the present study was to examine stability of
Department of Medicinal Chemistry, Medical University
of Lublin, Jaczewskiego 4, 20‑090 Lublin, Poland dihydralazine and hydrochlorothiazide, as individuals and
2 in the mixture, to detect such interactions using HPLC-UV,
Department of Pharmacognosy with Medicinal Plant Unit,
Medical University of Lublin, Chodźki 1, 20‑093 Lublin, LC-DAD and LC–MS methods.
Poland

13
Vol.:(0123456789)
1148 A. Gumieniczek et al.

Only few chromatographic procedures (HPLC) have degradation. The next step was to elucidate the possible deg-
been reported so far for determination of dihydralazine radation pattern of these drugs using LC-DAD and LC–MS
alone [3] or in presence of other drugs like hydrochlorothi- methods.
azide, triamteren and clonidine [4, 5]. In the study of Raul The results reported here can be useful in development
et al. [3], dihydralazine was determined on a C18 column of a new combined formulation of dihydralazine and hydro-
with a mobile phase consisted of phosphate buffer of pH chlorothiazide, by knowing dangerous conditions for both
3.0 and acetonitrile, and UV detection at 305 nm. How- constituents. In addition, these data may be the starting point
ever, there is no any report concerning forced degradation for further studies on new degradation products in terms of
of dihydralazine. In consequence, kinetics of degradation their potential toxicity.
as well as characterization of degradation products were
not reported at all.
More HPLC methods were elaborated for determination Experimental
of hydrochlorothiazide as an individual analyte [6, 7] or in
presence of other drugs like sartans [8–10], angiotensin-con- Materials and Methods
verting enzyme inhibitors [11–13], beta blockers [14–16]
and calcium channel blockers [17, 18]. Some HPLC methods Materials
were described as stability-indicating procedures capable to
determine hydrochlorothiazide in presence of its degradation Pharmaceutical grade (Eur. Ph.) dihydralazine sulfate and
products [19–26]. hydrochlorothiazide, and tetrabutylammonium hydrogen
In the study of Che [4], determination of dihydralazine sulfate for analysis from Sigma-Aldrich (St. Louis, MO,
and hydrochlorothiazide was performed using a CN column, USA), ammonium formate, formic acid, acetonitrile and
while a mobile phase consisted of acetonitrile and sodium methanol for LC from Merck (Darmstadt, Germany), acetic
heptane sulphonate. Two different wavelengths were used for acid ­(CH3COOH), sodium acetate ­(CH3COONa), hydro-
UV detection, i.e., 310 and 267 nm, for dihydralazine and chloric acid, sodium chloride (NaCl), sodium tetraborate
hydrochlorothiazide, respectively. Simultaneous determina- ­(Na2B4O7), sulphuric acid, sodium hydrogen phosphate
tion of dihydralazine and hydrochlorothiazide was elabo- ­(NaHPO4), sodium hydroxide (NaOH), kalium dihydrogen
rated by Jin et al. [5] using gradient elution on a C18 col- phosphate ­(KH2PO4) and kalium hydroxide for analysis from
umn. Acetonitrile and phosphate buffer of pH 3.0 containing POCh (Gliwice, Poland), acetonitrile and water for LC–MS
sodium heptane sulphonate were used as mobile phases A from J.T. Baker (Center Valley, PA, USA), ­Dihydralazinum®
and B, respectively. From these reports, it was clearly seen tablets 25 mg from Pabianickie Zakłady Farmaceutyczne
that simultaneous determination of these two drugs present- (Pabianice, Poland) and ­Hydrochlorothiazidum® tablets
ing different chemical properties, could be a big analytical 25 mg from Polpharma (Starogard Gdanski, Poland) were
challenge. used. Acetate buffer was prepared with 0.2 M C ­ H3COOH
Stability of hydrochlorothiazide in a solid state was tested and 0.2 M C­ H3COONa. Phosphate buffer was prepared with
at high temperature in the range 60–110 °C [19, 20, 22–25, 0.067 M ­KH2PO4 and 0.067 M ­Na2HPO4. Borate buffer was
27]. As far as photostability is concerned, experiments were prepared with 0.05 M ­Na2B4O7 and 0.1 M NaOH. Buff-
conducted at one wavelength (254 or 256 nm) [20, 24, 25] ers were prepared as described in Eur. Ph [2]. Buffers for
or in the entire UV range [24, 28–30]. They were carried kinetic studies have the same ionic strength of 1 M which
out in a solid state [20, 22, 24, 28, 31] and less frequently was attained with 4 M NaCl. The pH measurements were
in solutions [25, 29, 30]. Stability of hydrochlorothiazide done with a pH-meter HI9024C from Hanna Instruments
was studied in 1M HCl [19–22, 25, 30–32], 5 M HCl [28], (Padova, Italy).
1 M NaOH [19–22, 25, 27, 31–37], 5 M NaOH [28], buffers
[23] and methanol [19, 25]. One study on degradation of HPLC‑UV Method
hydrochlorothiazide by LC/MS method was reported in the
literature [27]. However, kinetics of degradation was studied Chromatographic Conditions
incidentally and only scarce information in this area was
found in the literature [23]. Analysis was performed with a model 306 pump with a loop
Thus, a new quantitative HPLC method for simultane- Rheodyne (20 µL) and a model UV170 detector controlled
ous determination of dihydralazine and hydrochlorothiazide by Omnic software from Gilson (Middleton, WI, USA).
in presence of their degradation products was elaborated Separation was carried out on a ­LiChrospher®CN column
and validated. Then, the concentration of non-degraded (125 × 4.0 mm, 5 µm) from Merck. The column was housed
dihydralazine and hydrochlorothiazide as a function of in a column heater set at 25 °C. The mobile phase consisted
degradation time was investigated, to determine kinetics of of water, 0.02 M tetrabutylammonium hydrogen sulfate

13
Kinetics and Characterization of Degradation Products of Dihydralazine and… 1149

and acetonitrile (20:65:15, v/v/v) adjusted to pH 3.5 with the drugs in the range from 20 to 120 µg mL−1. Then, six
0.5 M sulphuric acid. The flow rate of the mobile phase was injections were made onto the column for each concentra-
1.4 ml mL−1 while the detection was done at 235 nm. tion. The peak areas were plotted against the corresponding
concentration of the drugs to construct the calibration equa-
tions. The limit of detection (LOD) and the limit of quantifi-
Robustness cation (LOQ) were determined from the standard deviation
of the intercepts and slopes of the calibration lines at low
Small changes of analytical conditions, i.e., acetonitrile con- concentrations, using 3.3 and 10 factors for LOD and LOQ,
tent in a mobile phase (15 ± 2), pH (3.5 ± 0.1 unit), flow rate respectively.
of the mobile phase (1.4 ± 0.2 ml mL−1), detection wave-
length (235 ± 3 nm) and column temperature (25 ± 2 °C)
were made to study robustness of the developed method. Precision and Accuracy
One factor was changed at a time. For each combination,
three injections were carried out, using a working solution Precision was determined by analyzing the working solu-
containing 70 µg mL−1 of dihydralazine and hydrochloro- tions containing 30, 70 and 110 µg mL−1 of dihydralazine
thiazide. Finally, robustness of the method was expressed in and hydrochlorothiazide, three times during the same day
the forms of asymmetry factors and peak areas. and then on three subsequent days. Accuracy was estimated
by determining both active substances in six model mix-
tures and comparing the determined amounts to the nominal
Linearity values. The weighed portions of powdered tablets contain-
ing 25 mg of dihydralazine and 25 mg hydrochlorothiazide
Stock solutions of dihydralazine and hydrochlorothiazide were transferred to 25 ml volumetric flasks with ca. 15 mL
(1 mg mL−1) were used to obtain the working solutions of of methanol, sonicated for 30 min, diluted to the mark and

Fig. 1  Chromatograms HPLC-UV: a dihydralazine (4) and hydrochlorothiazide (3) in the calibration solutions; b dihydralazine (8) and hydro-
chlorothiazide (5) in the presence of their degradation products (3, 4, 6, 7)

13
1150 A. Gumieniczek et al.

filtered by nylon membrane filters (0.45 µm). Then, 0.6 mL in a climate chamber KBF P240 from Binder (Neckarsulm,
volumes were diluted to 10 mL and analyzed by the HPLC Germany) at 70 °C and 80% RH for 2 months. After forced
method described above. The assay was repeated six times, degradation, 10 mg of individual substances or 20 mg of the
individually weighing the respective tablet powders. mixture were weighed and dissolved with methanol to obtain
The concentrations of dihydralazine or hydrochlorothi- solutions of concentration 1.0 mg mL−1. After diluting with
azide were calculated using respective calibration equations methanol to cover the linearity range, the solutions were ana-
and expressed as RSD for precision and percentage recovery lyzed by our HPLC-UV method. The procedure was repeated
for accuracy. three times for each sample, and the concentrations of dihy-
dralazine or hydrochlorothiazide remaining after degradation
Degradation in a Solid State were calculated from the linear calibration equations.

Solid mixture containing dihydralazine and hydrochlorothi-

azide was prepared by weighing equal amounts of individual
substances and mixing them thoroughly in an agate mor-
tar. Individual substances and their mixture were placed in
standardized small flat vessels, so that the thickness of the
layer was approximately 3 mm. The samples were placed

Table 1  Validation of HPLC-UV method for simultaneous determi-

nation of dihydralazine and hydrochlorothiazide
Parameter Dihydralazine Hydrochlorothiazide
−1
Linearity range (µg mL ) 20–120 20–120
Slope 344,398 271,311
SD of slope 3134 1361
Intercept − 2,333,333 − 127,834
SD for intercept 471,404 74,342
R2 0.99886 0.99895
SD of R2 0.00053 0.00032
LOD (µg mL−1) 4.52 0.90
LOQ (µg mL−1) 13.69 2.74
Accuracy (% recovery) 99.24–101.03 99.45–101.54
Precision (RSD)
Intra-day 0.59–1.16 0.47–1.72
Inter-day 0.90–1.07 0.81–1.57
Retention time (min) 13.21 2.73
Asymmetry factor 1.5 1.0

Table 2  Percentage level of degradation of dihydralazine and hydro-

chlorothiazide under high temperature/humidity and under UV/Vis
light
Stress conditions Level of degradation (%)
Dihydralazine Hydrochlorothiazide
Individual Mixture Individual Mixture

70 °C/80% RH 22.00 29.70 12.83 17.72

1 ICH 2.85 28.98 16.71 19.93
3 ICH 13.26 59.58 27.78 33.99 Fig. 2  a UV/Vis spectra of dihydralazine, b hydrochlorothiazide
6 ICH 100.0 100.0 66.92 89.42 and c their mixture after photodegradation study: black line—stand-
ard (non-stressed sample), blue line—sample under 1 ICH dose, red
1 ICH = 18,902 kJ m2−1, 3 ICH = 56,706 kJ m2−1; 6 line—sample under 3 ICH doses and violet line—sample after 6 ICH
ICH = 113,412 kJ m2−1 doses

13
Kinetics and Characterization of Degradation Products of Dihydralazine and… 1151

Degradation in a Liquid State After diluting with methanol to covering the linearity range,
each sample was analyzed using our HPLC-UV method. The
Photodegradation procedure was repeated three times for each sample, and the
concentrations of non-degraded dihydralazine or hydrochloro-
Equal volumes (2 mL) of the stock solutions of dihydrala- thiazide were calculated from the linear calibration equations.
zine or hydrochlorothiazide (4 mg mL−1) were dispensed to When the level of degradation was at least 30% during
quartz glass-stoppered dishes (individually stressed drugs). 300 min, kinetic parameters were calculated. The logarithm
Equal volumes (1 mL) of the stock solutions of dihydralazine of the concentration of non-degraded substance was plot-
or hydrochlorothiazide (8 mg mL−1) were mixed in quartz ted against time of degradation to determine the order of
glass-stoppered dishes to obtain the mixtures. The samples reactions. The equations y = ax + b and R2 coefficients were
were placed in a Suntest CPS Plus chamber from Atlas (Lin- obtained from which further kinetic parameters, i.e., degra-
sengericht, Germany) and exposed to UV/Vis light in the dation rate constant (k) and degradation time of 50% sub-
range 300–800 nm, with energy equal to 18,902, 56,706 stance (t0.5) were calculated.
and 113,412 kJ m2−1. After forced degradation, the solu-
tions were diluted with methanol to cover the linearity range
and analyzed by our HPLC-UV method. The procedure was LC‑DAD and LC–MS Methods
repeated three times for each sample, and the concentrations
of non-degraded dihydralazine or hydrochlorothiazide were Before analysis, the buffers were removed from the samples
calculated from the linear calibration equations. using Bakerbond SPE C8 disposable extraction columns
(3 mL) from J.T. Baker and an UCT positive pressure Mani-
Kinetics fold station (Horsham, PA, USA). The ions were removed
from the bed with water, and then substances of interest were
From the stock solutions of dihydralazine or hydrochlorothi- eluted with methanol. Respective fractions were pooled and
azide (4 mg mL− 1), 1 mL volumes were dispensed to small dried under vacuum. Before LC analysis, they were recon-
glass tubes from Medlab (Raszyn, Poland) (individually stituted with acetonitrile.
stressed drugs). From the stock solutions of dihydralazine or The samples were analyzed with a 6530B accurate-
hydrochlorothiazide (8 mg mL−1), 0.5 mL volumes were dis- mass-QTOF-MS spectrometer with a dual ESI-Jet Stream
pensed in a similar way and mixed together. To each tube, 1 mL ion source, using an Eclipse XDB C18 (150 × 4.6 mm,
of appropriate stressor (1 M HCl, 1 M NaOH, buffers of pH 3.5 µm) column from Agilent Technologies (Santa Clara,
4, 7 and 10) was added. The tubes were tightly closed with CA, USA). The chromatograph was equipped with a DAD,
stoppers and placed in a thermostated water bath from WSL an autosampler, a binary gradient pump, and a column oven.
(Warszawa, Poland) at 80 °C. The samples were removed from Acetonitrile–water (1:99, v/v) with 10 mM ammonium for-
the bath after subsequently 15, 30, 45, 60, 75, 90, 105, 120, 135, mate (0.1%) (solvent A) and acetonitrile–water (95:5, v/v)
150, 165, 180, 195, 210, 225, 240, 255, 270, 285 and 300 min. with 10 mM ammonium formate (0.1%) (solvent B) were
They were immediately cooled and neutralized if necessary. used as mobile phases. The following elution procedure was

Table 3  Percentage level Stress conditions Degradation after Linear equation, y = ax + b R2 k ­(s−1) t0.5 (h)
of degradation and kinetic 300 min (%)
parameters of degradation of
dihydralazine in solutions Dihydralazine
1 M HCl 1.61 nc nc nc nc
Buffer pH 4 5.50 nc nc nc nc
Buffer pH 7 38.54 y = − 0.0018x + 4.7378 0.8820 2.70 × 10−5 7.13
Buffer pH 10 74.98 y = − 0.0043x + 4.7449 0.9665 7.70 × 10− 5 2.50
1 M NaOH 100.0 y = − 0.0184x + 4.0556 0.9989 5.09 × 10−4 0.38
Dihydralazine in the mixture with hydrochlorothiazide
1 M HCl 51.06 y = − 0.0064x + 4.9381 0.9871 3.97 × 10−5 4.85
Buffer pH 4 82.39 y = − 0.0064x + 4.9381 0.9871 9.65 × 10−5 1.99
Buffer pH 7 100.0 y = − 0.0080x + 4.4918 0.8243 3.28 × 10−4 1.50
Buffer pH 10 100.0 y = − 0.0092x + 4.7455 0.9805 3.53 × 10−4 1.26
1 M NaOH 100.0 y = − 0.0047x + 4.6211 0.9866 9.02 × 10−5 2.13

nc non-calculated

13
1152 A. Gumieniczek et al.

Table 4  Percentage level Stress conditions Degradation after Linear equation, y = ax + b R2 k ­(s−1) t0.5 (h)
of degradation and kinetic 300 min (%)
parameters of degradation of
hydrochlorothiazide in solutions Hydrochlorothiazide
1 M HCl 52.29 y = − 0.0018x + 4.5315 0.8117 3.11 × 10−5 4.68
Buffer pH 4 23.64 y = − 0.0007x + 4.7441 0.9366 1.50 × 10−5 12.83
Buffer pH 7 36.34 y = − 0.0011x + 4.6498 0.9642 2.50 × 10−5 7.67
Buffer pH 10 36.99 y = − 0.0012x + 4.7004 0.9506 2.51 × 10−5 7.49
1 M NaOH 37.97 y = − 0.0006x + 4.7518 0.9781 1.29 × 10−5 15.91
Hydrochlorothiazide in the mixture with dihydralazine
1 M HCl 53.98 y = − 0.0019x + 4.5717 0.9862 4.31 × 10−5 4.47
Buffer pH 4 70.71 y = − 0.0038x + 4.5422 0.9391 6.79 × 10−5 2.82
Buffer pH 7 93.72 y = − 0.0096x + 4.4038 0.9143 1.54 × 10−4 1.25
Buffer pH 10 94.14 y = − 0.0081x + 4.6094 0.9190 1.58 × 10−4 1.22
1 M NaOH 47.56 y = − 0.0017x + 4.7004 0.9438 3.59 × 10−5 5.36

a Fig. 4  Fragmentation pattern of NH2

dihydralazine in negative ioni- N
5.00
zation mode: CID off set 10V
4.50 N
y = -0.0011x + 4.6498
lnC of hydrochlorothiazide

4.00 R² = 0.9642 NH

3.50
N
NH2
3.00 y = -0.0096x + 4.4038 C8H9N6-
R² = 0.9143 Exact Mass: 189,09
2.50

2.00

1.50
0 50 100 150 200 250 300 N
Time [min]
NH
Hydrochlorothiazide Hydrochlorothiazide + dihydralazine
b
5.00
N
NH2
4.50 C8H9N4-
Exact Mass: 161,08
4.00 y = -0.0018x + 4.7378
R² = 0.8820
lnC of dihydralazine

3.50

3.00
CH3
2.50 y = -0.0080x + 4.4918
R² = 0.8243
2.00

1.50 N
0 50 100 150 200 250 300
C8H8N-
Time [min]
Exact Mass: 118,07
Dihydralazine Dihydralazine + hydrochlorothiazide

Fig. 3  First-order plots of drugs degradation in the buffer of pH 7: adopted: 0–60 min, 0–95% of solvent B with a stable flow
a dihydralazine degraded as an individual and in the mixture with
rate 0.4 mL min−1. The injection volume for the samples was
hydrochlorothiazide; b hydrochlorothiazide degraded as an individual
and in the mixture with dihydralazine. Errors bars on the curves rep- 10 µL. The analysis was conducted at 25 °C.
resent the SD values of triplicate samples LC/MS analysis was performed according to the fol-
lowing parameters of the ion source: a negative ion mode
(–ESI), gas (­ N2) flow rate 12 L min−1, nebulizer pressure

13
Kinetics and Characterization of Degradation Products of Dihydralazine and… 1153

35 psig, vaporizer temperature 350 °C, sheath gas tempera- hydrochlorothiazide in presence of their degradation prod-
ture 400 °C, sheath gas (­ N2) flow 12 L min−1, m/z range ucts. It is worth mentioning that similar reports concerning
100–1000 mass units with an acquisition mode auto MS/MS, dihydralazine were not found in the literature.
collision induced dissociation (CID) 10 and 40 eV with MS At the beginning, several trials were carried out to obtain
scan rate of 1 spectrum per s and 2 spectra per cycle, VCap good resolution between the drugs and their degrada-
4000 V, skimmer 65 V, fragmentor 150 V and Octopole RF tion products. They involved the use of different columns,
Peak 750 V. Additionally, the analysis was made in auto MS/ mobile phases and flow rates. First, C8 and C18 columns
MS with excluded m/z at 966.0007 and 112.9856 for nega- (125 × 4.0 mm, 5 µm) were used and the mixtures contain-
tive ion mode, corresponding to the m/z of reference ions. ing water, methanol and acetonitrile as mobile phases. As
The obtained experimental mass values were used to gen- far as C8 column was concerned, any mixture and any flow
erate molecular formula of the products, on which basis their rate of the mobile phase (0.8–1.8 mL min−1) did not result in
structures were predicted. The most postulated structures sufficient separation of hydrochlorothiazide and its degrada-
were justified through the mechanisms of their formation. tion products. All peaks of interest showed retention times
below 2 min. When a C18 column was used, it was showed
that content of acetonitrile in the mobile phase could not
Results and Discussion exceed 20% to obtain symmetrical peaks of hydrochlorothi-
azide. However, its retention time was still very short and
Development and Validation of LC‑UV Method resolution between the peaks of degradation products was
not achieved. Thus, addition of ion pair reagents was decided
A simple, isocratic HPLC-UV method was developed and one of them, i.e., tetrabutylammonium hydrogen sulfate
for simultaneous determination of dihydralazine and was effective in good separation of the peaks of interest.

Fig. 5  a Chromatogram DAD of dihydralazine (1) and its degradation product (2) in 1M NaOH. b Negative ion ESI LC/MS of degradation
product of dihydralazine (1,4-dihydrazinylidenophthalazine); CID off set 10 V

13
1154 A. Gumieniczek et al.

However, retention of dihydralazine was too strong in these The method was found to be linear over the concen-
conditions (retention times above 20 min). Therefore, a CN tration range of 20–120 µg mL −1 for both drugs, with
column was tried together with mobile phases containing average R 2 of 0.9989 for dihydralazine and 0.9990 for
not more than 20% of acetonitrile. It was showed that the hydrochlorothiazide. The calculated LOD and LOQ were
retention time of hydrochlorothiazide was still very short 4.52 and 13.69 µg mL−1 for dihydralazine, and 0.90 and
while that of dihydralazine was very long. Thus, acetonitrile 2.74 µg mL−1 for hydrochlorothiazide. The RSD values in
content was decreased to 15% and ion pair reagent (tetrabu- the range 0.59–1.16% for dihydralazine and 0.47–1.72%
tylammonium hydrogen sulphate) was added. As a result, the for hydrochlorothiazide (1-day precision), and 0.90–1.07%
retention time of hydrochlorothiazide was extended to ca. for dihydralazine and 0.81–1.57% for hydrochlorothiazide
3 min, while that of dihydralazine shortened to ca. 13 min, (inter-day precision) were obtained. Accuracy of the
maintaining an acceptable shape of the peaks. A huge differ- method was confirmed by determining both drugs in pow-
ence in retention times of dihydralazine and hydrochlorothi- dered tablets. Recovery values were obtained in the range
azide was still observed but separation of all peaks of inter- 99.24–100.99% for dihydralazine and 99.45–101.54%
est was possible. Respective chromatograms showed that the for hydrochlorothiazide (Table 1). The chromatograms
peaks of dihydralazine and hydrochlorothiazide were free obtained for the samples of powdered tablets showed the
from interferences of the degradation products, confirming peaks of interest free from interferences of excipients, con-
selectivity of the method (Fig. 1). firming selectivity of the method.
Resistance to small changes in analytical parameters,
i.e., acetonitrile content (15 ± 2%), pH (3.5 ± 0.1 unit), Degradation in a Solid State
flow rate (1.4 ± 0.2 ml mL −1 ), detection wavelength
(235 ± 3 nm) and column temperature (25 ± 2 °C) was Our study showed that dihydralazine was sensitive to
examined. Uniformity of the obtained peak areas con- high temperature and humidity (22.01% degradation after
firmed the robustness of the method. However, the calcu- 2 months at 70 °C/80% RH). In the mixture with hydrochloro-
lated values of peak symmetry indicated sensitivity of the thiazide, there was further increase of degradation to 29.70%
method to changes of detection wavelength and flow rate (Table 2). Therefore, it could be concluded that degradation
of the mobile phase. of dihydralazine was accelerated by hydrochlorothiazide.

Table 5  The identified degradation products of dihydralazine and hydrochlorothiazide

Dihydralazine [M-H]- Hydrochlorothiazide [M-H]- Mixture
m/z m/z

O O
NH O O
S S
NH 161.04 H2N NH 293.94 m/z 275.98

N Cl N
NH2

Impurity C Impurity A

O O
O O O O O O
S S S
121.03 283.96 S
H2N NH2 H2N NH

HN
NH2 N
NH2 Cl

O
Phenylmethylhydrazine Impurity B 2H-1,2,4-Benzo
thiadiazinesulfonamide
1,1,4-trioxide
NH2 O O
N O O
S S
H2N NH
N 187.04 259.98

N N

N
NH2

1,4-Dihydrazinylideno 1,2,4-Benzothiadiazine-7-
phthalazine sulfonamide 1,1-dioxide

13
Kinetics and Characterization of Degradation Products of Dihydralazine and… 1155

In the literature, a similar effect of hydrochlorothiazide was 2.85%. However, under higher exposures, its degradation
documented for its solid mixture with cilazapril [38]. was 13.26% (56,706 kJ m2−1 or 3 ICH doses) and 100%
After 2 months at 70 °C/80% RH, hydrochlorothiazide (113,412 kJ m2−1 or 6 ICH doses). In addition, its UV/Vis
as individual showed degradation equal to 12.83%. Accord- spectrum changed after these expositions (Fig. 2a). Dihy-
ing to the literature, the highest degradation of hydrochlo- dralazine mixed with hydrochlorothiazide underwent higher
rothiazide (ca. 15%) occurred at 100 °C (after 5 h) [25] and photodegradation than an individual substance. Under
65 °C (after 24 h) [26]. In the mixture with dihydralazine, energy equal 1 ICH dose, the level of degradation rose to
an increase of hydrochlorothiazide degradation to 17.72% 28.98% (Table 2). These data allowed to draw the conclusion
was observed (Table 2). that hydrochlorothiazide increased sensitivity of dihydrala-
These data allowed to draw the conclusion on mutual zine to light. It was also observed as changes in respective
influence of both dihydralazine and hydrochlorothiazide UV/Vis spectra (Fig. 2c). To the best of our knowledge, the
on their stability. As a consequence, the need to adequately results presented here are the first in the area of photostabil-
protect these active substances from high temperature and ity of dihydralazine.
humidity was clearly shown. Present experiment showed that hydrochlorothiazide was
sensitive to light (16.71% degradation upon energy equiva-
Photodegradation lent to 1 ICH dose) (Table 2). According to the literature,
similar level of degradation (ca. 18%) was observed under
In our study, the energy of 18,902 kJ m2−1 or 1 ICH dose monochromatic light at 256 nm [25]. However, much lower
was equivalent to 1,200,000 lx h and 200 W m2−1 [39]. photodegradation (0.1–5.5%) was also reported [29, 30].
Dihydralazine fulfilled the requirements of the test confirm- In our experiment, the influence of light was confirmed by
ing photostability, because its percentage degradation was changes in the UV/Vis spectra of the stressed samples. After

Fig. 6  a Chromatogram DAD of dihydralazine (1) and its degradation products (2, 3) under UV/Vis light. b Negative ion ESI LC/MS of degra-
dation product of dihydralazine (2): CID of set 10 V

13
1156 A. Gumieniczek et al.

Fig. 7  Proposed degradation NH2

pathway of dihydralazine in 1 M N
NaOH and under UV/Vis light

N
NH

N
NH2
C8H9N6-
Exact Mass: 189,09

NH2
N
N
N
NH
N

N
NH2 N
C 8H 9 N 4 - NH
C8H7N6-
Exact Mass: 161,04
Exact Mass: 187,04
HN
NH
C7H9N2-
Exact Mass: 121,03

O O O O
O O O O
S O O
S S
H2N N -HCN S S
H2N -SO2 HN

Cl N Cl Cl
H NH2 NH2
C7H7ClN3O4S2- C6H6ClN2O4S2- C6H6ClN2O2S-
Exact Mass: 295,96 Exact Mass: 268,95 Exact Mass: 204,98

Fig. 8  Fragmentation pattern of hydrochlorothiazide in negative ionization mode: CID off set 10 V

6 ICH doses, a clear shifting of one of the absorption maxi- Degradation in a Liquid State
mum of hydrochlorothiazide was observed (Fig. 1b). It con-
firmed photosensitivity of hydrochlorothiazide and indicated The present study proved stability of dihydralazine in a
the need to apply appropriate photoprotection for pure drug strongly acidic environment (1.61% of degradation in 1 M
and its formulations. HCl after 300 min). On the contrary, the highest decom-
Hydrochlorothiazide mixed with dihydralazine was more position was observed in a strongly alkaline environment
degraded in comparison with an individual substance (19.93 (100% of degradation in 1 M NaOH). In addition, the sub-
vs. 16.71%, 33.99 vs. 27.78% and 89.42 vs. 66.92% after stance degraded in buffers of pH 4, 7 and 10 (5.50, 38.54
1, 3 and 6 ICH doses, respectively) (Table 2). The above and 74.98% of degradation, respectively). Dihydralazine in
data as well as respective UV/Vis spectra confirmed the a mixture was degraded to a much greater extent. The great-
mutual influence of both components on their photostabil- est increase of degradation occurred in the buffer of pH 7
ity (Fig. 2c). (100 vs. 38.54%). In addition, a high level of degradation

13
Kinetics and Characterization of Degradation Products of Dihydralazine and… 1157

O O 20, 22, 25, 28, 30–32]. Hydrochlorothiazide was shown to

O O
S be stable in the buffer of pH 2.0 and less stable at pH 9.0,
S
H2N N where 9.07% of the drug degraded [23]. Our experiments
confirmed that hydrochlorothiazide was prone to degra-
N
dation in 1 M HCl (52.29%) and 1 M NaOH (37.97%).
Cl
H Degradation in buffers of pH 4, 7 and 10 was higher in
C7H7ClN3O4S2- comparison to Ref. 23 (23.64–36.99%), probably because
Exact Mass: 295,96
of using higher temperature. Degradation of hydrochloro-
thiazide was further intensified in the mixture with dihy-
O O dralazine. The greatest increase of degradation occurred in
O O the buffer of pH 7 (93.72 vs. 36.34%) (Table 4).
S S
HN NH2

Kinetics
Cl NH2
C6H7ClN3O4S2-
Exact Mass: 283,96 We completed our experiments by calculating the degrada-
tion kinetics of dihydralazine and hydrochlorothiazide. It
was found that degradation of dihydralazine, individually
stressed and in the mixture, proceeded as the first-order
O O reactions. Values of the rate constants (k) were at the levels
O of ­10−5–10−4 s−1. The shortest t0.5 (0.38 h) was calculated
S
HN
for 1M NaOH (Table 3). Hydrochlorothiazide degradation
S N
was also observed as the first-order reactions. Values of
O the observed k for individually stressed hydrochlorothi-
Cl NH2
C6H6ClN2O2S- NO2S- azide were at the level of 1­ 0−5 s−1. The shortest t0.5 was
Exact Mass: 204,98 Exact Mass: 77,97 obtained in 1 M HCl (4.68 h). In the mixture, the quickest
degradation of hydrochlorothiazide was calculated at pH
7 and 10 (k values at the level 1­ 0−4 s−1), where the lowest
values of t0.5 were obtained (1.22–1.25 h) (Table 4). In the
mixture, both the substances were observed to degrade
O O harder and faster in almost all experimental conditions.
S However, the biggest differences occurred in the buffer of
HN pH 7. Therefore, respective data were depicted as xy dia-
grams (Fig. 3). Due to the lack of other data in this area,
NH2 the results presented here are a valuable supplement to the
C 6H 5 N 2O 2S - literature resources.
Exact Mass: 169,01

Identification of Degradation Products

Fig. 9  Proposed degradation pathway of hydrochlorothiazide in solu-
tions
As a result of negative ionization of dihydralazine, a depro-
tonated molecule [M–H]− of m/z 189.09 was formed. Then,
(51.06%) was observed in 1 M HCl, where individually fragment ions of m/z 161.08 and 118.07 were produced.
stressed dihydralazine showed stability (Table 3). Based on these results, it was shown that dihydralazine was
According to the cited works, the highest degradation of fragmented by loss of hydrazine and breaking the phena-
hydrochlorothiazide occurred in methanol (53.56%) [25], zine ring (Fig. 4). The Eur. Ph. monograph of dihydralazine
1 M NaOH (69.60%) and 1 M HCl (81.70%) [21]. In other lists three impurities, A (4-hydrazinophthalazin-1-amine),
works, degradation in acidic medium ranged from 0.4 to B (hydrazine) and C (1-hydrazinophthalazine) [2]. In our
17.86% while in alkaline medium from 0.15 to 36.82% [19, LC-DAD and LC/MS studies, a product of m/z 187.04

13
1158 A. Gumieniczek et al.

Fig. 10  a Chromatogram DAD of hydrochlorothiazide (3) and its degradation products (1, 2) under UV/Vis light. b Negative ion ESI LC/MS of
degradation product of hydrochlorothiazide (1): CID off set 10V

was observed after degradation in 1 M NaOH. The respec- Ph. monograph of hydrochlorothiazide lists three impuri-
tive chromatogram and mass spectrum were presented in ties, A (6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide
Fig. 5. It was identified as 1,4-dihydrazinylidenophthalazine 1,1-dioxide, chlorothiazide), B (4-amino-6-chloroben-
(Table 5). Such a product was not described in the litera- zene-1,3-disulfonamide) and C (6-chloro-N-[(6-chloro-
ture so far. In the buffer of pH 10 and under UV/Vis light, 7-sulfamoyl-2,3-dihydro-4H-1,2,4-benzothiadiazin-4-yl
two degradation products were observed as fragment ions 1,1-dioxide)methyl]-3,4-dihydro-2H-1,2,4-benzothiadia-
of m/z 121.03 and 161.04 (Fig. 6). The first product was not zine-7-sulfonamide 1,1-dioxide) [2]. In the present study, a
described in the literature so far, while the second one was degradation product of hydrochlorothiazide was detected as
identified as impurity C (Table 5). The proposed degradation a fragment ion of m/z 283.96. It was identified as impurity
pathways for dihydralazine are presented in Fig. 7. B. It confirmed the results obtained by Belal et al. [22] and
Using a negative ionization mode, a deprotonated mol- Mahajan et al. [27] where the same product was detected as a
ecule [M–H]− of hydrochlorothiazide of m/z 295.06 was result of acid and alkaline hydrolysis of hydrochlorothiazide.
obtained from which, after losing the HCN molecule, an The structure of the resulting product indicated breaking of
ion of m/z 268.95 was formed. Further fragmentation led the 1,2,4-benzothiadiazine ring and loss of formaldehyde.
to formation of an ion of m/z 204.98 (loss of S ­ O2) (Fig. 8). The resulting product further fragmented yielding an ion
These results are consistent with previous results concern- of m/z 204.98 (loss of the sulfonamide group), from which
ing fragmentation of hydrochlorothiazide [27]. The Eur.

13
Kinetics and Characterization of Degradation Products of Dihydralazine and… 1159

O O O O (fragment ions of m/z 121.03, 169.09, 178.99, 187.04,

H2N
S S
N
204.98 and 293.94). In addition, further products
occurred after degradation in 1 M HCl (m/z 199.07), 1 M
Cl N
H
NaOH, buffers (m/z 211.07) and under UV/Vis light (m/z
C7H7ClN3O4S2- 169.05). Unfortunately, they were not identified by us.
Exact Mass: 295,96
However, quite a new product of photodegradation of
hydrochlorothiazide of m/z 275.98 was detected in the
O O O O mixture (Fig. 12) and identified as 2H-1,2,4-benzothiadi-
S
O
S
O
S
O O azinesulfonamide 1,1,4-trioxide (Table 5). This indicated
S
H2N N H2N N a new way of hydrochlorothiazide photodegradation by
detaching the chlorine atom and oxidation at the nitrogen
N
Cl
C7H5ClN3O4S2-
N
atom (Fig. 13).
C7H6N3O4S2-
Exact Mass: 293,94
Exact Mass: 259,98

Conclusions
O O Dihydralazine in a solid state was sensitive to high tem-
O S N
S N N perature and humidity, while in solutions it was degraded
O N
by UV/Vis light and pH below 4. Hydrochlorothiazide in a
NO2S-
Exact Mass: 77,97 C7H3N2O2S-
N
C7H3N2- solid state was sensitive to high temperature and humidity
Exact Mass: 178,99 Exact Mass: 115,03 as well as to UV/Vis light. In solutions, it degraded in 1M
HCl, 1M NaOH, buffers and under UV/Vis light. In the
Fig. 11  Proposed degradation pathway of hydrochlorothiazide under stressed individual samples of dihydralazine and hydro-
UV/Vis light chlorothiazide, (phenylmethyl)hydrazine and 1,2,4-benzo-
thiadiazine-7-sulfonamide 1,1-dioxide were observed for
the first time.
hydrogen chloride was detached to form an ion of m/z 169.9. Percentage degradation of drugs in the mixture was
The proposed fragmentation profile was drawn in Fig. 9. greater than of individual drugs under the same stress
Under UV/Vis light, two other fragment ions of m/z conditions. An interesting effect was observed for dihy-
values 293.94 and 259.98 were detected (Fig. 10). The dralazine which became sensitive to acidic degradation.
first was identified as chlorothiazide (impurity A) [2]. Increased sensitivity of both active substances to stress
The second product was not described in the literature so and potent interactions between them were confirmed by
far and was identified as 1,2,4-benzothiadiazine-7-sul- new degradation products detected, e.g., 2H-1,2,4-benzo-
fonamide 1,1-dioxide (Table 5). Based on these results, it thiadiazine-7-sulfonamide 1,1,4-trioxide.
was concluded that light facilitated detaching the chlorine The results presented here complemented the current
atom from the aromatic ring (Fig. 11). A similar result knowledge about degradation processes of dihydralazine
was observed and described for amiloride [40]. In the and hydrochlorothiazide. These data may be the starting
present experiment, chlorothiazide further fragmented point for further studies on new degradation products in
by losing the sulfonamide group and hydrogen chloride terms of their potential toxicity and then, for qualifying
yielding an ion of m/z 178.99. Previously, only chloro- them as new related substances in pharmacopoeial mono-
thiazide was described as a result of photodegradation of graphs. These data may also serve as a starting point for
the parent drug [27]. designing new two-component formulations.
Most of these degradation products were observed in
the mixture of dihydralazine and hydrochlorothiazide

13
1160 A. Gumieniczek et al.

Fig. 12  a Chromatogram DAD of hydrochlorothiazide (7) and degradation products of hydrochlorothiazide and dihydralazine (1–6, 8) in the
mixture under UV/Vis light. b Negative ion ESI LC/MS of degradation product of hydrochlorothiazide (2): CID off set 10 V

Fig. 13  Proposed photodeg- O O O O O O O O

radation pathway of hydro- S S
chlorothiazide in presence of S S
H2N N H2N N
dihydralazine

Cl N N
H
C7H7ClN3O4S2-
Exact Mass: 295,96
O
C 7 H 6 N3O5 S 2 -
Exact Mass: 275,98

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priate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
Conflict of Interest The authors declare that they have no conflict of
interest.

Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors. References

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