Article

pubs.acs.org/OPRD

Understanding and Control of Dimethyl Sulfate in a Manufacturing

Process: Kinetic Modeling of a Fischer Esterification Catalyzed
by H2SO4
John P. Guzowski, Jr.,*,† Edward J. Delaney,‡ Michael J. Humora,† Erwin Irdam,† William F. Kiesman,†
Albert Kwok,† and Amy D. Moran†
†
Biogen Idec, 14 Cambridge Center, Cambridge Massachusetts 02142, United States
‡
Reaction Science, 11 Deerpark Drive Suite 202, Monmouth Junction, New Jersey 08852, United States
*
S Supporting Information

ABSTRACT: The formation and fate of monomethyl sulfate

(MMS) and dimethyl sulfate (DMS) were studied by proton
NMR for a sulfuric acid catalyzed esterification reaction in
methanol. The kinetic rate constants for DMS and MMS were
determined at 65 °C by fitting time-dependent experimental
data to a model using DynoChem. In refluxing methanol, sulfuric
acid was converted to monomethyl sulfate (MMS) in nearly
quantitative yield within 45 min. Once formed, the MMS
underwent a reversible esterification reaction to form DMS.
Dimethylsulfate reacted with methanol to regenerate MMS and form dimethyl ether. A byproduct of the esterification reaction
was water, which further consumed DMS through hydrolysis. On the basis of derived rate constants, in refluxing methanol, DMS
would not be expected to exceed 4 ppm in the reaction mixture at equilibrium. In the presence of the carboxylic acid substrate,
DMS was not detected in the reaction mixture. The reaction pathways of this system have been systematically investigated, and
the results of this study will be presented.

■ INTRODUCTION
There is significant interest in the pharmaceutical manufactur-
the gentle reflux of H2SO4 in methanol encountered during the
esterification process used to make the active pharmaceutical
ing and regulatory communities regarding the generation and ingredient (API). The question posed by the regulator was
analysis of genotoxic impurities.1 In 2010, 26 articles were easily addressed with a direct analysis method using gas chro-
published that highlighted the phrase “genotoxic impurities” as matography (GC) to measure dimethyl sulfate in the isolated
a keyword.2 We received the following inquiry during regu- drug substance. Multiple batches produced at commercial scale
latory review of a commercial process to manufacture a drug were tested with a validated GC method (limit of detection,
candidate: “Based on the synthetic pathway, including puri- LOD = 0.1 ppm), and DMS was not observed in the API.
fication steps, of the drug substance, please address the potential From a quality-by-design (QbD) perspective, the more
formation of methyl hydrogen sulfate and dimethyl sulfate relevant technical question for the chemistry team was “could
impurities with a validated analytical method, which is sensitive DMS be generated in the reaction mixture prior to API
enough to detect and quantitate the potential genotoxic impu- isolation?”
rities. The maximum daily intake of the potential genotoxic The GC method used to analyze isolated drug substance
impurities should not exceed 1.5 μg per person per day by taking could not be applied to in-process solutions that contained sul-
the highest dose of your clinical candidate.” furic acid, carboxylic acid ester, and methanol. Detailed method
The process in question consisted of the sulfuric acid- development efforts revealed that DMS could actually be for-
catalyzed esterification of a dicarboxylic acid in methanol. A med in the heated injection port when samples of H2SO4 in
large body of literature describes dimethyl sulfate (DMS) as a methanol were subjected to GC analysis. It was not possible at
known genotoxin; furthermore, its reactivity as an electrophilic that time to successfully mitigate artifacts arising from the sample
methylating agent in SN2 alkylations is greater than that of matrix, and HPLC methods lacked the necessary sensitivity and
methyl iodide.3 In contrast, monomethyl sulfate (MMS) is a selectivity.
poor alkylating agent and is not genotoxic.3 The potential An orthogonal analytical method for DMS was also inves-
formation of DMS as an impurity in the reaction matrix thus tigated using a well-known derivatizing agent, triethylamine
became the primary focus of our investigation. (TEA), followed by HPLC−MS analysis.4 Unfortunately, it was
Commercial DMS manufacture is typically performed with
SO3 and anhydrous methanol catalyzed by Pd or other tran- Received: November 10, 2011
sition metals.3 These forcing conditions are quite different than Published: December 23, 2011

© 2011 American Chemical Society 232 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239
Organic Process Research & Development Article

discovered that the diester drug substance also reacted with A multistep reaction mechanism based on published work6 is
triethylamine, producing a false positive result for DMS. proposed in Scheme 1 for the generation and consumption of
Around this time, an investigation was published that MMS and DMS.
described the use of GC−MS to measure reaction kinetics
for the formation of methyl methanesulfonate.5 This compound Scheme 1. Proposed pathways for the formation and
is also a genotoxic impurity and of regulatory interest because degradation of DMS and MMS
of its potential to form under conditions used to manufacture
mesylate salts of an API.
Herein, we discuss the application of 1H NMR techniques
and kinetic modeling to the complex multistep equilibria
of dimethyl sulfate and monomethyl sulfate under reaction
conditions used to manufacture the drug substance.

■ RESULTS AND DISCUSSION

This investigation was undertaken to better understand the
potential to form sulfuric acid-derived impurities in a com-
mercial diester synthesis. The reaction employs methanol and In contrast to previously studied alkylsulfonic acids (such as
catalytic amounts of sulfuric acid to effect esterification of the methanesulfonic acid and ethanesulfonic acid), sulfuric acid
dicarboxylic acid starting material (eq 1). Sulfuric acid-derived
can undergo sequential reactions with methanol to generate
reaction byproduct could potentially include monomethyl sul-
two different sulfate esters. Monomethyl sulfate is a relatively
fate and dimethyl sulfate.
benign, nongenotoxic impurity that can be controlled like other
process-related contaminants.3 Conversely, DMS is a known
genotoxic species and must be controlled to very low levels.7
It was important to examine the formation and fate of these two
sulfate esters under actual process conditions to fully under-
stand the potential process risk presented by DMS.
The esterification reaction kinetics were first examined to In order to make the study of this complex multistep mech-
establish the baseline conditions needed to further study for- anism manageable, the pathway was broken into four discrete
mation of the two sulfate ester impurities (MMS, DMS). The steps, and the results of each were used to develop a compre-
reaction mixture consisted of 1 equiv dicarboxylic acid, hensive kinetic model. Where possible, a rate constant was
16.5 equiv methanol (5.7 volumes), and 0.25 equiv concentrated independently evaluated and determined. The effect of water
H2SO4 (0.12 volumes). The solution was heated to a gentle concentration on each corresponding rate constant was sepa-
reflux at 65 °C, and time-course samples were analyzed by 1H rately evaluated and incorporated into the final model. Results
NMR. The reaction was complete within 3 h, and integral peak from each step allowed an overall mechanistic model to be
data were used to produce the reaction kinetic profile presented constructed. Early kinetic studies were conducted without the
in Figure 1 (millimoles vs time). dicarboxylic acid substrate, but it was included in subsequent
investigations.
Monomethyl Sulfate: Rate of Formation and Degra-
dation. The initial sets of experiments were designed to inves-
tigate the formation (k1) and degradation (k−1) of monomethyl
sulfate, the key intermediate in the pathway to the formation of
dimethyl sulfate.
1. Formation of Monomethylsulfate (k1). The formation of
MMS is depicted in eq 2:

Dry methanol (<0.01% water) was mixed with concentrated,

dry sulfuric acid and heated to 65 °C. After waiting one minute
to reach temperature, seven discrete samples were collected
within 12 min and 1H NMR data (16 transients) obtained for
these solutions. Equilibrium was reached within 1 h, resulting in
Figure 1. Reaction profile for the esterification of a dicarboxylic acid an essentially quantitative conversion of H2SO4 into mono-
by methanol and sulfuric acid as measured by NMR. Experimental data
represented by individual points on the graph. methyl sulfate. The heated sample remained unchanged after
several days’ storage in a sealed tube. Identity of the MMS
resonance in the NMR spectrum was confirmed by spiking an
The NMR-derived data in Figure 1 were consistent with lab- authentic sample of monomethyl sulfate into the reaction mix-
and production-scale sampling studies where the esterification ture. The MMS peak integral (CH3, 3.45 ppm) was normalized
reaction was >95% complete within 1 h at 65 °C. Subsequent to the methyl peak resonance of the methanol solvent (CH3,
kinetic studies focused on generation and consumption of the 3.18 ppm) and DynoChem8 used to calculate the forward
two sulfate esters and completed within 60 min. rate constant (k1) from the peak-integral data. The study was
233 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239
Organic Process Research & Development Article

conducted twice with good agreement between the two derived 999:1 in favor of the forward reaction, thus conservatively
rate constants. The experimental (diamonds) and fitted (line) defining k−1 as 6.5 × 10−8 L/mol s.
data for the formation of monomethyl sulfate are presented in In related work, Wolfenden and Yuan9 measured the rate
panel a of Figure 2 (early time points in reaction) and panel b constants for the hydrolysis of MMS in water over a range of
temperatures and pH and found the extrapolated value (at 25 °C)
to be 1.7 × 10−8 L/mol s (1 M HCl, T = 40−100 °C).
Results from both laboratories agree that MMS is rapidly
formed and stable over a wide range of temperatures and water
concentrations. These results are also consistent with the fact
that monomethyl sulfuric acid is a relatively poor alkylating
agent.
Dimethyl Sulfate: Rate of Degradation and Forma-
tion. Dimethylsulfate is formed and consumed in a complex set
of interrelated equilibria. Rates of DMS solvolysis (methanol-
ysis and hydrolysis) can be readily measured, and these are the
pathways that consume DMS. However, the amount of DMS
that is formed by the forward reaction between methanol and
MMS is very small. Hence, to simplify the experimental design,
the DMS methanolysis and hydrolysis rates were empirically
measured (eqs 4 and 5, respectively).

The equilibrium level of remaining dimethylsulfate following

methanolysis was then used to derive the forward rate of for-
mation for DMS.
1. Methanolysis of DMS (k3 ). Solutions of 1.5 mol % DMS
in methanol were heated in a sealed tube to 35 °C, and time-
dependent NMR spectra were collected for these mixtures.10
Data obtained over 60 min showed that dimethyl ether (DME)
and MMS resonances gradually increased over time with a
Figure 2. Experimental and predicted fit of data for the formation of concurrent decline in the DMS signal. Representative 1H NMR
monomethyl sulfate at 65 °C. (a) Data points collected in the first spectra from these experiments are presented in Figure 3. The
12 min, 0.379 M H2SO4. (b) Equilibrium data points obtained from
15 to 180 min, 0.365 M H2SO4.

of Figure 2 (later reaction time points). The H2SO4 concen-

trations were similar for both studies but they were run at
different scales (5 mL vs 50 mL). The second-order forward
rate constant (k1) for the formation of MMS at 65 °C was
experimentally determined to be 6.5 × 10−5 L/mol s with a
confidence interval of ±7% RSD.
2. Hydrolysis of Monomethylsulfate (k−1). As illustrated in
Scheme 1, monomethyl sulfate can either hydrolyze back to
sulfuric acid (k−1) or further react with methanol to form DMS
(k2). The hydrolysis of MMS is presented in eq 3

Figure 3. Time-resolved spectra for the methanolysis of DMS at 35 °C.

chemical shift of dimethyl ether was comparable to that

The rate constant of this reaction was measured by spiking reported in the literature and used to confirm its identity.11 Use
water into solutions containing MMS (1.5 mol %) and of NMR data helped verify the dynamic reaction pathways
monitoring the heated, sealed vessel by 1H NMR for 45 h. proposed in Scheme 1.
Water was spiked into the matrix at two different levels, −6 and The gradual downfield shift of the exchangeable OH reso-
12 mol %. In both cases, the MMS level remained virtually nance (4.8 ppm) is consistent with the presence of a strong acid
unchanged, confirming that the equilibrium for this reaction lies (i.e., MMS) as outlined in eq 4.
far to the right (eq 3). In order to develop the larger model, the A duplicate set of experiments were performed under typical
MMS equilibrium constant K = (k1/k−1) was assigned a value of reaction conditions at 65 °C (again spiking 1.5 mol % DMS
234 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239
Organic Process Research & Development Article

into dry methanol with <0.01% w/w H2O and no sulfuric acid)
and monitoring the reaction for 60 min. The reaction profile
for the methanolysis of DMS (k3) is presented in Figure 4. The

Figure 4. Experimental reaction profiles for the methanolysis of DMS

at 65 °C for two replicate experiments. The DMS concentration was
measured after mixing dimethylsulfate with dry methanol.

diamonds in Figure 4 represent two replicate measurements

used by DynoChem to generate the second-order rate constant.
This reaction was assumed to be essentially irreversible under
nominal plant process conditions since the reaction vessels are
not pressurized and the resultant DME would bubble out of
solution.
The rate constant for the methanolysis of DMS (k3) was
derived from this experimental data using DynoChem and found
to be 3.1 × 10−5 L/mol s ±10% RSD.
The rate measured by Kolesnikov and co-workers12 for this
reaction was reported as 2.27 × 10−5 s−1 at T = 35 °C.
Kolesnikov also presented the Arrhenius parameters for this
system, and they can be used to calculate the rate constant at
65 °C, which yields a value of 1.4 × 10−5 L/mol s (methanol =
23.4 M) and is comparable to the rate determined in this study.
2. Hydrolysis of DMS (k−2). The hydrolysis rate of dimethyl
sulfate was determined by spiking known amounts of water into
a mixture of DMS and methanol at three different levels −14.3,
18.1, and 21.9 mol %. The mol % of water was calculated as
[100(mol H2O/total moles in mixture)]. The reactions were
monitored for 60 min at 65 °C, and DMS peak integrals were
obtained from the NMR data. Time-dependent reaction pro- Figure 5. (a) Experimental reaction profiles for the hydrolysis of DMS
files for the consumption of DMS are presented in Figure 5 as at 65 °C, water spike −14.3 mol %. (b) Experimental reaction profiles
the following: (a) 14.3 mol %, (b) 18.1 mol %, and (c) 21.9 mol %. for the hydrolysis of DMS at 65 °C, water spike −18.1 mol %. (c)
The predicted DMS content (mmol) in the presence of Experimental reaction profiles for the hydrolysis of DMS at 65 °C,
different water spikes was performed using the k2 (hydrolysis) water spike −21.9 mol %.
and k3 (methanolysis) rate constants.
The rate constant for the hydrolysis of DMS (k−2) was Profiles of the DMS methanolysis and hydrolysis experi-
derived by fitting all of the data to a DynoChem model that ments revealed that MMS was formed more rapidly when the
employed the time-dependent peak integral data presented methanol solution contained water, whereas dimethylether
above (Figure 5 a−c). The experimentally determined value for formation was retarded by the addition of H2O. These results
k−2 was found to be 1.3 × 10−4 L/mol s ±20% RSD. suggest a bimolecular mechanism for DMS methanolysis (k3)
Kolensikov13 reported a value of 6.14 × 10−4 s−1 (T = 35 °C) and hydrolysis (k−2). Methanol and water compete to consume
for this reaction along with the Arrhenius parameters that were the available DMS in an SN2-like displacement reaction. The
then used to determine the second-order rate constant at more nucleophilic water molecule is able to hydrolyze DMS
55 °C, which was the upper temperature limit of their study. The faster than dimethylsulfate can react with methanol (k−2 > k3).
rate constant adjusted for water concentration (H2O = 54.8 M) However, in this reaction system, methanol is in much greater
was 7.86 × 10−5 L/mol s and is in good agreement with the abundance and will be the primary species that consumes DMS.
result from this study. These two competing reactions are presented in Scheme 2.
235 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239
Organic Process Research & Development Article

Scheme 2. Competition between methanol and water for DMS

Teasdale et al. described similar findings in their study of The DMS peak was measured against the MMS 13C satellite
the methanolysis and hydrolysis of methyl methanesulfonate.5 peak because it more closely approximated the dimethyl sulfate
Their investigation using O18 labeled methanol confirmed that signal level and would better reflect the true solution concen-
oxygen in the dimethyl ether, formed during methanolysis of tration. The natural abundance of the 13C isotope was 1.11%
methyl methanesulfonate, came from methanol and not meth- and equally divided between two symmetrical resonances.
anesulfonic acid. When corrections were applied for the number of chemically
3. Formation of Dimethylsulfate (k2). The formation of equivalent protons in DMS (6 protons) and MMS (3 protons)
DMS from MMS can be expressed as eq 6: the steady-state DMS concentration in the reaction matrix
was calculated to be 157 ppm relative to MMS,16 resulting in a
derived k2 rate constant of 4.9 × 10−9 L/mol s.
The forward rate constant for methyl methanesulfonate for-
mation, a closely related chemical system, has previously been
Using the mechanistic pathway presented in Scheme 1, the reported by Teasdale5 to be 7.1 × 10−8 s−1. In that case, the rate
forward reaction rate for DMS (k2) can be calculated under constant was measured directly and assumed to be dependent
steady-state conditions as follows: upon conversion of a single species (methanol protonated by
methanesulfonic acid) into methyl methanesulfonate and water.
(a) The rate of formation and loss of DMS are in balance The rate constant was therefore expressed as a first-order process.
d[DMS]
=0 forward rate = k[CH3SO3−MeOH2+]
dt
= k 2[MMS][CH3OH] − k−2[DMS] In the current study, a second-order forward rate constant is
reported since the measurement was carried out indirectly from
[H2O] − k3[DMS][CH3OH] a methanolysis reaction at equilibrium. However, a more direct
comparison can be made. Since the equilibrium for the
(b) Solving for k2 protonation of methanol by methyl methanesulfonic acid lies
[DMS](k−2[H2O] + k3[CH3OH]) far to the right, the rate can also expressed as a second-order
k2 = rate constant by employing the alternative convention used in
[MMS][CH3OH] this work:
(c) Under dry conditions, the equation can be further simplified forward rate = k 2[CH3SO3H][MeOH]
k [DMS][CH3OH] [DMS]
k2 = 3 = k3 By expanding the kinetic pre-equilibration step to include
[MMS][CH3OH] [MMS] the methanol concentration term (23.1 M concentration of
methanol in the earlier work employing 1 M methanesulfonic
Thus, the rate constant for the formation of DMS (k2) can acid in methanol), the first-order rate constant can be trans-
be calculated if [DMS] can be measured at steady state formed into an equivalent second-order rate constant of 3.1 ×
since k3, [MMS], and [CH3OH] have been experimentally 10−9 L/mol s. The DMS formation rate constant reported here
determined. (4.9 × 10−9 L/mol s) is only slightly higher than the published
In early attempts to measure DMS under typical esterifi- forward rate of methyl methanesulfonate formation.5
cation conditions, the dimethyl sulfate resonance was not ob- The rate constants for the entire reaction system are sum-
served, likely because the concentration was too low (ppm) to marized in Table 1, as determined using the DynoChem soft-
be detected by NMR.14 Therefore, a H2SO4 solution was pre- ware kinetics module.
pared in large excess relative to the standard process conditions Comparison of DynoChem Kinetic Model to Measured
(0.075 mol equivalent H2SO4 relative to methanol or a 5-fold DMS Concentrations in a Process Stream. The derived
rates constants resulted from statistical fitting of all data to a
concentration increase) and heated for an extended time period
kinetic model that was generated to predict the dynamics of
(7 h) at 65 °C. Under these forcing conditions, a small, but DMS formation/degradation. The model was subsequently
clearly discernible peak was observed at a chemical shift of used to study the formation of DMS in a simulated esterifica-
3.7 ppmthe DMS resonance region. An authentic sample of tion process stream and demonstrated that steady-state levels of
DMS spiked into this mixture confirmed the peak’s identity dimethylsulfate were formed within the typical 3-h operating
(15). The DMS peak integral area was approximately 5.7% of window employed in the plant. Assuming anhydrous starting
the upfield 13C satellite peak of MMS. conditions, the steady-state concentration of DMS in the reaction
236 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239
Organic Process Research & Development Article

Table 1. Rate constants for the diesterification process

1
Assigned to be 999× slower than k1 for purpose of model creation based on experimental data. 2Confidence interval was not determined as data
obtained directly from NMR experiment (not DynoChem) in a single measurement, value derived from experimental data collected for other rate
constant.

vessel was predicted to not exceed 4 ppm in the reaction mix- The API was also tested for DMS, and it was not detected in
ture.17 Typically, reagent grade methanol used in the manu- the drug substance (<0.1 ppm, the method’s limit of detection).
facturing plant will not be anhydrous and adventitious water
would even further suppress the formation of DMS in this
reaction system because the rate of hydrolysis (k−2) is 5 orders
of magnitude faster than formation of the DMS (k2). However,
this calculation can be used as the worst case scenario in a
failure-mode evaluation of the process. Dimethyl sulfate that spiked into the reactor during these
The chemical systems initially used to predict the rate constants experiments exceeded the level predicted by the kinetic model
did not contain the carboxylic acids that would be present during (4 ppm in solution) by 250-fold. These results confirm that the
drug substance manufacture. Dimethyl sulfate is a strong alkylating process conditions actually control the in situ level of generated
agent and might also react with the carboxylic acid substrate, DMS. One possible explanation is the reaction between DMS
further reducing the DMS concentration in the mixture. Teasdale and carboxylic acid substrate, which is present in large excess
previously reported5 that sulfonate ester formation was effectively (3700×).18 This reaction is illustrated as eq 7:
quenched when the reaction mixture contained a weak base in The effective consumption of DMS in the reaction matrix
slight excess, which was used to simulate a drug substance. ensures that the level of this impurity does not approach the
threshold for toxicological concern.
An in-process reaction mixture containing all components
was tested for DMS content by GC−MS and the result was
<0.5 μg/mL (ppm) as presented in Table 2, entry 1. The DMS
concentration in the reaction solution was below the predicted value
■ CONCLUSION
The formation and fate of monomethyl sulfate and dimethyl
from the kinetic model and confirms that dimethyl sulfate was not sulfate were studied by proton NMR for a methanol−sulfuric
generated at measurable levels under actual process conditions. acid esterification reaction, and these results have been pre-
To further challenge the reaction system, a DMS stock solu- sented. The rate constants were determined using DynoChem to
tion was gravimetrically prepared and an aliquot spiked into the model the time-dependent peak integral data collected from a
reaction mixture at a concentration of 1000 ppm. Samples were series of experiments probing each mechanistic step. Sulfuric
taken during the course of reaction and solutions analyzed for acid is converted into MMS in near quantitative yield in less
DMS using a modified GC−MS sample extraction preparation than 60 min under reaction conditions. Monomethyl sulfate
procedure. Dimethyl sulfate was present at 20 ppm after 1 h is the more persistent of these two species and appears quite
and quickly decreased to <0.5 ppm after only 3 h (Table 2). stable under process conditions. Dimethyl sulfate rapidly con-
verts into MMS and is readily hydrolyzed by water. The pre-
Table 2. Analysis of DMS in reaction solutions by GC−MS sence of adventitious water further serves to reduce DMS in the
using a liquid−liquid extraction procedure reaction mixture and retards its formation. Under typical pro-
cess conditions, the amount of dimethylsulfate present in the
DMS spiked in DMS measured in reaction solution is predicted to be less than 4 ppm. To date,
experimental reaction mixture reaction mixture
entry conditions (μg/mL, ppm) (μg/mL, ppm) DMS has not been detected in any drug substance batches
1 T = 0 h (analysis 0 <0.5
(<0.1 ppm, LOD).
before DMS spike)
2

3
T = 1 h (250× excess
DMS spike)
T = 3 h (250× excess
1000

1000
20

<0.5
■ EXPERIMENTAL SECTION
NMR was chosen as the preferred method for this study, owing
DMS spike) to its directness of measurement for all species, its wide dynamic
237 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239
Organic Process Research & Development Article

range of measurement, and the fact that mechanistic assumptions (100-g batch size). The effective DMS solution concentration
could be confirmed under the same set of conditions that rate was 1000 ppm. The following conditions were used for this
constants were being measured. spiking experiment:
All NMR spectra were obtained in, and referenced against, 1 Charge 100 g of dicarboxylic acid starting material.
DMSO-d6 at 2.5 ppm using a Varian 500 MHz NMR. Concen- 2 Charge 580 mL of methanol and start agitation at 480 rpm.
trated sulfuric acid, dicarboxylic acid (starting material), and 3 Charge 21.3 g of sulfuric acid
dimethyl ester (product) were obtained from the Sigma-Aldrich 4 Heat the reaction to 65 ± 1 °C.
Chemical Co. Authentic standards of MMS (Na salt) and DMS 5 Charge DMS, ∼0.58 g, accurately weighed (0.55 wt %/wt,
were also obtained from Sigma-Aldrich. For spiking identification or 5500 ppm, relative to the resulting API).
purposes, an authentic a sample of MMS was prepared by pass- 6 Maintain at this temperature for 3 h. Remove aliquots for
ing a methanolic solution of the commercially available sodium analysis during 3 h reaction time.
salt through Amberlite FPA 22 resin (H form, 10 mol equiv), 7 Cool to 22 °C in 8 h and hold for 2 h.
and concentrating to an oil on a flash evaporator. 8 Filter and wash cake four times with 70 mL of methanol.
All small-scale reactions performed in the study were mag- 9 Dry the cake at 22 °C at 100 mmHg.
netically stirred and carried out using an insulated oil bath 10 Sample dry API for DMS content.
maintained at 65 ± 1 °C. Methanol used in this study con-
tained no more than 0.01% water, and was further dried using 3 Å
zeolite molecular sieves that had been predried overnight at
175 °C. Concentrated sulfuric acid, purchased from Sigma-Aldrich,
■
*
ASSOCIATED CONTENT
S Supporting Information

was >99.9% nominal purity and was used as provided. Experimental data and DynoChem results from the modeling
Sampling of reactions involved removal of a minimum of studies. This material is available free of charge via the Internet
150−400 μL of the reaction mixture and addition to DMSO-d6 at http://pubs.acs.org
(lock solvent). The final volume in the NMR sample tube was
650 μL. Samples were chilled in an ice/water bath, and ana-
lyzed within 5−10 min of their preparation. Reaction profiles
■ AUTHOR INFORMATION
Corresponding Author
were tracked by plotting reaction completion (via reactant inte- [email protected].
gral measurements) against time.
Although DMS was present in only trace amounts upon
reaching equilibrium following extended methanolysis, the S/N
ratio of the DMS peak was >10:1 and thus could be measured
■ REFERENCES
(1) (a) Robinson, D. I. Org. Process Res. Dev. 2010, 14, 946−959. (b)
Genotoxic Impurities- Strategies for Identification and Control; Teasdale,
with confidence. However, MMS and its left satellite peak (nearby A., Ed.; John Wiley & Sons: NJ, 2010.
in chemical shift to the DMS resonance) could not be accurately (2) Number of citations for genotoxic impurity studies for the year
integrated electronically due to the baseline deflections in this 2010 as obtained from a Scifinder search.
region from the broad methyl resonance of methanol (solvent). (3) (a) Friedli, F. E. In Rate of Quaternization as a Function of
As a result, the spectrum was enlarged, and the peaks were the Alkylating Agent. World Conference on Oleochemicals into the
physically extracted and weighed in order to obtain the molar 21st Century: Proceedings ; World Conference on Oleochemicals − Into
ratio. The error associated with this measurement procedure was the 21st Century, Kuala Lumpur, Malaysia, October 8−12, 1990;
expected to be no more than 10% and comparable to other Applewhite, T. H., Ed.; American Oil Chemists’ Society: Champaign,
IL, 1990; pp 296−297. (b) Thayer, F. J. Am. Chem. Soc. 1924, 46,
sources of experimental and computational uncertainties.
1044−1046. (c) Tan, E.; Brimer, P.; Schenley, R.; Hsie, A. J. Toxicol.
Fitting of all experimental data to generate the rate constant Environ. Health 1983, 11, 373−380.
data and an overall kinetic model was carried out using DynoChem (4) An, J.; Sun, M.; Bai, L.; Chen, T.; Liu, D. Q.; Kord, A. J. Pharm.
(version 3.3). Biomed. Anal. 2008, 48, 1006−1010.
A modified GC−MS method (liquid−liquid extraction) was (5) Teasdale, A.; Delaney, E. J.; Eyley, S. C.; Jacq, K.; Taylor-Worth,
developed to analyse the in-process solutions and mitigate K.; Lipczynski, A.; Hoffmann, W.; Reif, V.; Elder, D. P.; Facchine,
artifacts related to the sample matrix. The extraction method K. L.; Golec, S.; Oestrich, R. S.; Sandra, P.; David, F. Org. Process Res.
was also used to analyse isolated drug substance development Dev. 2010, 14, 999−1007.
samples. An Agilent 6890N GC was fitted with a Supelco (6) Teasdale, A.; Eyley, S. C.; Delaney, E.; Jacq, K.; Taylor-Worth, K.;
Equity-1701 (30 m long × 0.32 mm ID, 1.0 μm film thickness) Lipczynski, A.; Reif, V; Elder, D.; Facchine, K. L.; Golec, S.; Oestrich,
R; Sandra, P.; David, F. Org. Process Res. Dev. 2009, 13, 429−433.
column using helium carrier gas at 2 mL/min (constant flow). (7) Sobol, Z.; Engel, M.; Rubitski, E; Ku, W. W.; Aubrecht, J.;
Analyte detection was accomplished by an FID. The column Schiestl, R. Mutat. Res. 2007, 633, 80−94. (b) International Agency for
was heated from 50 °C to 280 °C over 21 min. Approximately Research on Cancer (IARC) Monographs Program on the Evaluation of
∼100 mg of the sample was accurately weighed into a 10 mL Carcinogenic Risks to Humans (2004) for Dimethyl Sulfate; International
conical tube, and 10.0 mL of 0.1 M NaCl was added to the Agency for Research on Cancer: Lyon, France, 1999; Vol. 71, pp 575−
sample container and vortexed for 5 min. Then, 1.0 mL of 588.
methyl tert-butyl ether (MBTE) was added to the mixture and (8) DynoChem, version 3.3; DynoChem, Inc.: Wilmington, DE,
vortexed for another 5 min to extract the DMS. The solution 19803.
was centrifuged at 4000 rpm for 10 min, and 200 μL of the (9) Wolfenden, R; Yuan, Y. Proc. Natl. Acad. Sci U.S.A. 2007, 104,
83−86.
MTBE top layer was removed for analysis by GC.
(10) Dimethyl ether (DME) resonances were not observed when this
DMS Spiking Experiment. A stock solution of DMS was study was first attempted at 65 °C in a standard NMR tube.
prepared and used to challenge the disposition of dimethylsul- (11) Nagai, Y.; Matubayasi, N.; Nakahara, M. J. Phys. Chem. A 2005,
fate under process conditions (Table 2). The final concen- 109, 3558−3564.
tration of DMS in the reactor was 0.55% (5500 ppm), relative (12) Kolensikov, V. A.; Efremov, R. V. Kinetika I Kataliz 1979, 20,
to API, and spiked into the mixture at the start of the reaction 813.

238 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239

Organic Process Research & Development Article

(13) Kolensikov, V. A.; Efremov, R. V.; Danov, S. M.; Gryaznova,

L. V. Kinetika I Kataliz 1977, 18, 1065−1066.
(14) It was also possible that the weak DMS signal (CH3OSO2O-
H3C, 3.7 ppm) was obscured by the presence of resonances arising
from the reaction products. The monoester and diester products yield
13
C satellite signals that reside in the same spectral region as DMS.
(15) A second minor peak was observed at 3.8 ppm and later iden-
tified as a solvent-related impurity arising from the methanol used in
this study.
(16) [DMS] = (1H peak area DMS/1H peak area from total MMS) ×
(equivalent 1H for MMS)/(equivalent 1H for DMS) × (13C abundance
for MMS) = 0.057(3/6) × 0.0055 = 157 ppm.
(17) Assuming virtually anhydrous starting conditions, 2.39 × 10−5
moles of DMS will be present in the reaction mixture under steady-
state conditions as predicted by DynoChem. The DMS amount can be
normalized to the dicarboxylic starting material (1 mol) to yield a
value of 24 ppm expressed as (mol DMS/mol starting material). The
effective DMS solution concentration was determined to be 4 ppm by
correcting for the reaction volume (∼6 vols).
(18) The steady-state concentration of methyl ester intermediate was
determined by DynoChem to be 8.97 × 10−2 mol and compared to that
of DMS (2.39 × 10−5 mol) to determine molar excess (3700×).

239 dx.doi.org/10.1021/op200323j | Org. ProcessRes. Dev. 2012, 16, 232−239