Modeling of the Photodecomposition
Products of Ranitidine Hydrochloride
With N, N’ – Dimethylnitroacetamidine.
Christopher Downing
Submitted: December 23, 2003
College of Saint Benedict and Saint John’s University, Minnesota 56321
Abstract
The
direct
photodecomposition
products
of
Ranitidine
Hydrochloride is modeled using N, N’ – dimethylnitroacetamidine.
The resulting products are isolated using High Pressure Liquid
Chromatography, LC. Spectral Data is gathered using a Mass
Spectrometer detector on the LC. The Isolated peaks will be tested
to be reproducible.
The model compound, N, N’ –
Dimethylnitroacetamidine,
exhibits
some
of
the
same
characteristics as the Ranitidine Hydrochloride in the LC and
LC/MS data. Both have a product that has the same starting
weight, but different ion fragmentation, UV absorbencies, and is
more polar. This indicates a possible rearrangement.
Introduction
The intent of this research is to determine the photodecomposition
products of N, N’ – Dimethylnitroacetamidine using LC/MS. N, N’ –
Dimethylnitroacetamidine models the moiety of Ranitidine Hydrochloride that
undergoes direct photolysis. The kinetics, direct and indirect photolysis
information combined lead to the selection of this model compound. 1
With the knowledge of the products formed by the photodecomposition of
N, N’ – Dimethylnitroacetamidine it is hoped to find similarities in the
decomposition of Ranitidine Hydrochloride, and thus determine the
photodecomposition products.
Direct photolysis occurs when the compound being studied directly
absorbs the UV light and then undergoes a reaction, resulting either in a
rearrangement, or the loss of a functional group.
Ranitidine Hydrochloride is an H-2 receptor antagonist.2 Ranitidine blocks
histamine in the stomach, interfering with the production of stomach acid.3 One
commercial name for Ranitidine is Zantac®. The gastrointestinal pharmaceutical
may not be completely metabolized by the body resulting in the excretion of
some Ranitidine.
It is important to understand the environmental fate of pharmaceuticals
and the broader group of Pharmaceuticals and Personal Care Products, further
known as PPCPs. PPCPs travel from the household, industry, and water runoff
into water treatment plants, but may not be biodegraded, and appear in holding
waters. 4 This may be a major environmental pollutant, due to the consistent use
of PPCPs.
Results
Figure 1.
Figure 2.
Figure 3.
N, N’ – Dimethylnitroacetamidine - Photodecomposition
RT: 0.00 - 10.00
NL:
3.63E4
To tal Scan
P DA m a ms
44_0
uAU
4.49
20000
0.04
uAU
0
NL:
2.50E4
To tal Scan
P DA m a ms 1
6.46
20000
10000
2.58
0.02
0
3.97
NL:
2.12E4
To tal Scan
P DA m a ms 2
6.51
uAU
20000
10000
2.57
3.97
0.04
0
7.76
NL:
2.16E4
To tal Scan
P DA m a ms 3
6.48
uAU
20000
10000
2.57
0.12
uAU
0
uAU
3.09 3.30
3.97
4.41
7.79
5.38 5.72 5.94
8.47 8.86 9.20 9.54 9.92
NL:
1.95E4
To tal Scan
P DA m a ms 4
6.46
2.57
10000
0.52 0.89 1.27 1.64 1.95 2.18
0
3.25
3.97 4.32
7.74
5.33 5.55
NL:
1.48E4
To tal Scan
P DA m a ms 6
6.48
2.59
10000
3.11 3.31
0.20
0
uAU
1.73 2.12
3.97
4.40
7.77
5.32 5.67 5.87
8.22 8.45 8.77
9.43 9.92
NL:
1.24E4
To tal Scan
P DA M A M S
8
2.59
6.45
10000
5000
0.07
0
2.13
3.11 3.43
2
3
0.98 1.45 1.66
0
1
3.96
4.34
4
5.50
7.77
5.91
5
Time (min)
6
7
8.60 8.80
8
9.33
9
9.79
10
Figure 4.
83.0
100
M A MS_MS
8_030729085346#67 RT:
1.44 AV: 1 NL: 2.16E6 T: +
c ESI Full ms2
132.00@30.00 [
50.00-500.00]
90
Relative Abundance
80
70
60
50
40
30
20
10
100.9
132.1 149.8 167.5
115.0
72.7
0
13
M A MS_MS
8_030729085346#190
RT: 4.11 AV: 1 NL: 2.74E5
T: + c ESI Full ms2
132.00@30.00 [
50.00-500.00]
10
8
86.0
71.0
6
4
100.9
2
149.8
131.9
167.8
70.2
0
50
100
150
200
250
300
m/z
350
400
450
500
Figure 5.
100
210.0
M A MS_MS
8_0307290853
46#381 RT:
1.27 AV: 1 NL:
3.65E5 microAU
90
Relative Absorbance
80
70
60
50
40
30
20
10
330.0
0
15
355.0
375.0
410.0
445.0 460.0 475.0
505.0 520.0 535.0 555.0
585.0
310.0
M A MS_MS
8_0307290853
46#1171 RT:
3.90 AV: 1 NL:
5.37E4 microAU
12
10
8
220.0
6
4
2
400.0 410.0
375.0
0
200
250
300
350
400
wavelength (nm)
445.0
475.0
505.0 520.0
450
540.0
500
575.0
550
595.0
600
Ranitidine Hydrochloride
Figure 6.
Ran Photo MS,MS#120
RT: 2.58 AV: 1 NL:
3.89E5 T: + c ESI Full
ms2 315.10@35.00 [
85.00-500.00]
270.0
100
90
Relative Abundance
80
70
60
50
187.9
40
30
124.1
20
10
117.1
128.7 170.0 176.1
0
77
215.0
211.0
239.0 256.1
284.9
270.0
Ran Photo MS,MS#360
RT: 7.78 AV: 1 NL:
2.99E5 T: + c ESI Full
ms2 315.10@35.00 [
85.00-500.00]
60
176.0
50
40
224.0
30
124.1
20
215.0
10
144.0
170.0
110.0
191.0
241.1
0
100
150
200
250
271.1
300
m/z
350
400
450
500
Figure 7.
225.0
100
Ran Photo
MS,MS#645
RT: 2.15 AV:
1 NL: 1.58E4
microAU
90
Relative Absorbance
80
70
60
50
40
30
20
10
405.0
0
26
465.0
425.0 440.0
515.0
230.0
Ran Photo
MS,MS#2286
RT: 7.62 AV:
1 NL: 4.03E3
microAU
315.0
20
205.0
15
10
5
400.0 410.0
435.0 445.0
0
200
250
300
350
400
wavelength (nm)
450
500
550
600
N, N’ – Dimethylnitroacetamidine – Separation
Figure 8.
10 cm Discovery RP Amide C16 Column (Supelco)
100% Acetic Acid Buffer – pH 3.6
600 micro liters / min
RT: 0.00 - 10.00
NL:
1.05E5
3.68
100000
Total Scan
PDA M A
conc 2
10_2
90000
80000
70000
uAU
60000
50000
40000
30000
20000
10000
2.07 2.32
0.69
0
0
2.50
1.44 1.93
1
2
4.35 4.66
3
4
5
Time (min)
5.75 6.11
6
6.99 7.28 7.90 8.49
7
8
9.53 9.99
9
10
Figure 9.
10 cm Discovery RP Amide C16 Column (Supelco)
100% Acetic Acid Buffer – pH 3.6
400 micro liters / min
RT: 0.00 - 20.00
NL:
7.84E5
5.08
3.55
Total Scan
PDA MA
Con Sep
4ml_min
700000
600000
3.13
uAU
500000
400000
4.05
300000
200000
7.00
100000
7.53
1.51 2.64
0
0
2
4
6
8
9.05 10.02
10
Time (min)
11.75
13.43 14.28
12
14
16.50
16
18.44
18
20
Figure 10.
10 cm Discovery RP Amide C16 Column (Supelco)
100% Acetic Acid Buffer – pH 3.6
400 micro liters / min
RT: 0.00 - 20.00
NL:
7.81E5
9.98
Total Scan
PDA MA
Con
2ml_min 2
700000
6.67
6.24
600000
6.91
uAU
500000
400000
8.05
300000
13.47
8.35
200000
100000
14.33
16.83
0.12
0
0
2
4
6
8
10
Time (min)
12
14
16
18.53
18
20
Figure 11.
Data from 100% Buffer – Acetic Acid – pH 3.6
400 micro liters/min
15 cm Discovery RP Amide C16 Column (Supleco)
RT: 0.00 - 15.00
NL:
7.58E5
11.58
Total Scan
PDA MA
Conc Trial
2
700000
600000
7.57
500000
uAU
6.94
400000
300000
8.22
9.13
200000
10.31
100000
0.74 1.40
0
0
2.32 3.05
1
2
4.08 4.66
3
4
5
13.88
5.80
6
7
8
Time (min)
9
10
11
12
13
14
15
Ranitidine
Figure 12.
15 cm Discovery RP Amide C16 Column (Supelco)
100% Acetic Acid Buffer
400 micro liters / min
RT: 0.00 - 15.00
NL:
5.85E4
8.49
10.89
55000
Total Scan
PDA Ran
Conc Trial
1
50000
45000
40000
uAU
35000
30000
25000
20000
11.90
15000
10000
7.22 7.65
4.03
5000
10.43
0
0
1
2
3
4
5
6
7
8
Time (min)
9
10
11
12
13
14
15
Discussion
The structure of Ranitidine HCl and N,N’-dimethylnitroacetamidine, the
model compound, are represented in Figures 1 and 2, respectively. The model
compound has the same functional group as the end of Ranitidine HCl that
undergoes direct photolysis.
An understanding of the photodecomposition of the model compound can
be found in Figure 3, which shows the PDA detector information for seven
sample times. The model compound peak is at 6.50 min, and the most
significant decomposition peak is at 2.60 min, using the 10 cm RP Amide C16
column. Mass Spectrometry indicates that the major species at these two peaks
have the same mass-charge ratio, of 132. This is the molecular weight of the
model compound. Figure 4 shows that even though they have the same ratio,
they have different fragmentation patterns, the second fragmentation is for the
model compound, and matches the standard. As well the UV/Vis absorption is
different for the two molecules, and you can see this in Figure 5, the second
chromatogram is that of the model.
Ranitidine HCl shows similar characteristics, including the two different
peaks having the same mass-charge ratio, 315. Figure 6 shows the
fragmentation patterns for both peaks, the decomposition product is first. As well
Figure 7 shows the same type of difference in the UV/Vis absorption as the
model compound had, the bottom graph belongs to Ranitidine, the second peak
studied.
The samples were concentrated to 1.5 mL and prepared for the same
LC/MS sequence. Figure 8 through Figure 10 show the difference effected on
the separation and resolution of the model compound using different flow rates
with the same isocratic method on the 10 cm RP Amide C16 column. Figures 9
and 10 show little difference in the separation at a flow rate of 400 L/min
compared to 200 L/min. Figure 11 indicates that best separation currently,
though resolution is still poor, using a flow rate of 400 L/min on the 15 cm RP
Amide C16 column. It has been observed that there are multiple peaks in the
UV/Vis PDA detector output that are close, and had before concentration been
seen as only one peak. Effecting better resolution would allow each peak to be
assigned a mass-charge ration, then fragmentation patterns, and then hopefully
the fully elucidated structure.
Figure 12 indicates the separation of the Ranitidine HCl photodecomposition products run on the 15 cm column, using the isocratic Acetic Acid buffer.
It appears that Ranitidine HCl does not have this close packing of the
decomposition products, as it occurred in the model compound. Resolution does
need to be increased to positively identify each peak as being a single species.
This is why separation is so important, as the sample enters the mass
spec it is hard to assign mass-charge ratios if the resolution is not good, and
MS/MS fragmentation patterns will be impossible to assign.
Conclusion
It Can be concluded that the photodecomposition products of N, N’ –
Dimethylnitroacetamidine model the products of Ranitidine Hydrochloride, under
the same conditions. Future work will include determining what the structure of
the products of the model compounds photolysis are. And then apply that
information to the products of Ranitidine HCl and determine if a similar structure
does actually occur. This will be completed with more fraction collections.
One possible Product is the rearrangement of the model compounds nitro
group to form the following molecule. This arrangement is more polar, may lose
the N=O on the end, and fits the Mass Spec Data correctly. The total loss of the
nitro group also fits some of the other spectral data collected.
Being that the Mass Spec data indicates numerous mass fragments
occurring at the same time, this is investigated and these peaks are the same
daughter ion peaks of the known compounds, and occur because of the high
temperature of the ESI Capillary tube.
Experimental Section:
General Procedure:
All High Pressure Liquid Chromatography, further referred to as LC, was
completed with a Thermo Finnigan Surveyor® LC System. The LC was equipped
with a Photodiode Array detector, PDA, and Mass Spectrometer detector, MS.
The MS detector is a Thermo Finnigan LCQ Advantage, and uses a quadrupole
ion trap. The MS was equipped with an Electrospray Ionization source, ESI. 5
A buffer solution consisting of 850 µL of Glacial Acetic Acid and .250 g of
Ammonium Acetate diluted to 1000 mL with HPLC grade H2O. The pH was
balanced at 3.60, using NaOH or Acetic Acid if necessary. This buffer solution is
used on the LC as the mobile phase. The method that was determined most
effective at separation and resolution of the peaks is an isocratic 100 percent
acetate buffer. A previous buffer made with 700 µL concentrated Formic Acid
diluted to 1000 mL with HPLC grade H2O and balanced at a pH of 3.00 was
discontinued after an impurity was discovered in the Formic Acid.
The column used on the LC was a Discovery RP Amide C16 reverse
phase made by Supelco®. Two different columns were used one with length 10
cm by 4.6 cm and a longer column 15 cm by 4.6 cm.
Experimental Procedure:
Photolysis
The photolysis of both Ranitidine Hydrochloride and N, N’ –
Dimethylnitroacetamidine were accomplished using a set of stock solutions of
4 mM concentration, which was diluted ten times for a concentration of 400 µM.
The stock and diluted solutions were made using HPLC grade water. The diluted
samples were placed in Quartz test tubes and set on a rack designed to hold
them at a 45 degree angle to the sun. The samples were prepared in a dark
room, and covered with aluminum foil to avoid any extra photolysis. The test
tubes were then exposed to direct sunlight, and small samples were taken at
time zero, and subsequent hour and two hour time periods. These samples were
prepared for the LC/MS sequence.
The samples of Ranitidine HCl and model compound that underwent
photolysis each totaled about 250 mL. These samples were dried to a solid
using a SpeedVac system, over the course of 36 hours. The solid was then
redissolved in 1.5 mL of HPLC grade H2O to be used in the research of
separation and resolution methods.
Footnotes
1) Latch, Douglas E., Stender, Brian L., Packer, Jennifer L., Arnold, William A. and McNeill,
Kristopher. Photochemical Fate of Pharmaceuticals in the Environment: Cimetidine and
Ranitidine. Environ. Sci. Technol. 2003, 37, 3342-3350
2) Prescription List. <http://www.rxlist.com/cgi/generic/ranit.htm> August 2nd, 2003.
3) Medicine Net Product Information. <http://www.medicinenet.com/ranitidine/article.htm>
August 2nd, 2003.
4) Christian G. Daughton and Thomas A. Ternes. Pharmaceuticals and Personal Care
Products in the Environment: Agents of Subtle Change? Environmental Science Division,
U.S. Environmental Protection Agency and Institute for Water Research and Water Technology,
Wiesbaden-Schierstein Germany. 1991.
5) Thermo Finnigan Product Page. <http://www.thermofinnigan.com> August 4th, 2003.