Coupling and optimisation of online nuclear magnetic resonance spectroscopy and mass spectrometry for process monitoring to cover the broad range of process concentration

Article abstract of DOI:10.1002/mrc.4484

Real time online monitoring of chemical processes can be carried out by a number of analytical techniques, including optical and vibrational spectroscopies, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). As each technique has unique advantages and challenges, combinations are an attractive option. The combination of a 500-MHz ¹H NMR and a small footprint mass spectrometer to monitor a batch reaction at process concentration was investigated. The mass spectrometer was coupled into the flow path of an online reaction monitoring NMR. Reaction mixture was pumped from a 100-ml vessel to an NMR flow tube before returning to the vessel. Small aliquots were diverted into a sampling make-up flow using an active flow splitter and passed to the mass spectrometer. Advantages of the combination were observed. ¹H NMR was ideal for quantitation of high level components, whereas MS showed a greater capability for detecting those at low level. In preliminary experiments MS produced a limited linear relationship with concentration (0.02% to 2% relative concentration, 0.01 mg/ml–1.25 mg/ml), because of signal saturation at the higher concentrations. NMR was unable to detect components below 0.1% relative to concentration maximum. Optimisation of sample transfer to the MS extended the linearity to 10% relative to the concentration maximum. Therefore, the combination of online NMR and MS allows both qualitative and quantitative analysis of reaction components over the full process range. The application of the combination was demonstrated by monitoring a batch chemical reaction and this is described. Copyright

Full text of DOI:10.1002/mrc.4484

F
Coupling and Optimisation of On-Line Nuclear Magnetic Resonance Spectroscopy and
Mass Spectrometry for Process Monitoring to cover the broad range of process
concentration
Alexander Blanazs, Tony W. T. Bristow*, Steven R. Coombes, Tom Corry$, Mike Nunn and
Andrew D. Ray
Pharmaceutical Technology and Development, AstraZeneca, Silk Road Business Park, Charter
Way, Macclesfield, Cheshire, SK10 2NA.
*Corresponding author
$Current Address - School of Chemistry, The University of Manchester, Oxford Road,
Manchester, M13 9PL, United Kingdom.
Abstract
Real time on-line monitoring of chemical processes can be carried out by a number of analytical
techniques, including optical & vibrational spectroscopies, Nuclear Magnetic Resonance (NMR)
spectroscopy and Mass Spectrometry (MS). As each technique has unique advantages and
challenges, combinations are an attractive option.
The combination of a 500 MHz 1H NMR and a small footprint mass spectrometer to monitor a batch
reaction at process concentration was investigated. The mass spectrometer was coupled into the
flow path of an on-line reaction monitoring NMR. Reaction mixture was pumped from a 100 ml
vessel to an NMR flow tube before returning to the vessel. Small aliquots were diverted into a
sampling make-up flow using an active flow splitter and passed to the mass spectrometer.
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doi: 10.1002/mrc.4484
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1
Advantages of the combination were observed. H NMR was ideal for quantitation of high level
components, whereas MS showed a greater capability for detecting those at low level. In preliminary
experiments MS produced a limited linear relationship with concentration (0.02 % to 2 % relative
concentration, 0.01 mg/mL – 1.25 mg/mL), due to signal saturation at the higher concentrations.
NMR was unable to detect components below 0.1 % relative to concentration maximum.
Optimisation of sample transfer to the MS extended the linearity to 10 % relative to the
concentration maximum. Therefore, the combination of on-line NMR and MS allows both qualitative
and quantitative analysis of reaction components over the full process range. The application of the
combination was demonstrated by monitoring a batch chemical reaction and this is described.
Introduction
Direct on-line analysis of a chemical process is advantageous as this allows the progress to
be evaluated in real time, the point of completion to be established and impurity formation
to be understood.
Many analytical techniques have been employed and evaluated to monitor both batch and
continuous chemical processes. Examples include in-line UV absorption, infra-red (IR) and
Raman spectroscopies that employ in-line flow cells. [1-5] However it is not always possible to
identify intermediates and impurities from the IR data and IR may also lack sensitivity to
detect low level impurities. On-line reaction monitoring using high performance liquid
chromatography (HPLC) is another useful approach which has been demonstrated with the
application of a microflow HPLC for on-line monitoring of batch and continuous flow
reactors. [6,7]
High-field NMR is an attractive option to monitor a process from both a qualitative and
quantitative perspective and its application has been demonstrated for a pharmaceutical
process.[8] Coupling a high-field NMR spectrometer to a batch reaction is a considerable
challenge due to the size and requirements of a high-field spectrometer, along with issues
such as co-location. However, a number of options are available including dedicated flow
probes designed for on-flow applications or flow tube type solutions enabling the use of
standard 5 mm probes. Khajeh and co-workers have proposed an NMR flow-cell comprising
a standard 5 mm NMR tube for continuous process monitoring.[9] The flow-cell allows
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solution to be pumped continuously through the NMR tube and then returned to the
process vessel. The concentric design of the flow-cell minimises changes in magnetic field
homogeneity as the cylindrical symmetry is maintained. Commercial systems are also
available;[10] these benefit from the ability to acquire accurate kinetic data on-flow in
contrast to “in-tube” reactions as demonstrated by Foley et al.[11] In the same report, Foley
et al also presented evidence for the effect of mixing on the rate of reaction determined by
NMR, with the rate determined highly dependent on the NMR monitoring method used.[11]
A double chamber NMR tube has also been investigated by Mix and co-workers for
monitoring reactions conducted in a NMR tube.[12] The study concludes that a 3 mm glass
tube can be placed inside a 5 mm glass NMR tube with a small piece of silicone sealant, this
seal then creates two compartments inside the NMR tube. On removal of the inner tube
inside the NMR magnet using a rod, the reactants mix initiating the reaction. An outer
casing approach is suggested by Lindon and co-workers.[13] In a publication by Dalitz, the use
of non-deuterated solvents and the effect of flow rate on sample through the NMR flow-cell
was discussed.[14]
In an alternative pharmaceutical application, Coombes et al
demonstrated quantification of three active pharmaceutical ingredients simultaneously in
an on-line dissolution experiment, with an external standard approach used for
calibration.[15] Imine/aminal formation in a range of different solvents and catalysed
esterification reactions are other examples where synthetic reactions have been monitored
[16, 17]
quantitatively using high-field NMR.
Despite the challenges of coupling a high-field
spectrometer to a reaction vessel for on-line monitoring, greater selectivity over IR and UV
spectroscopies simplifies calibration and data analysis. Small footprint low-field NMR is also
an option for process monitoring due to the reduced cost and increased flexibility, and their
use has also been demonstrated. [18, 19]
Mass spectrometry (MS) has also been employed to monitor reactions in real time. MS can
have an advantage over other analytical techniques in that the specificity of the m/z of an
ion can be employed to confirm the formation of a proposed product and characterise
impurities and the presence of residual reactants. A key challenge issue for on-line MS is
the transfer of a sample from the process that is initially unsuitable for MS. This is due to
issues of high process concentration and possibly a reaction solvent that is not conducive to
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efficient ionisation of the components it contains. The sample must be representative of the
reaction whilst at the same time being compatible with the MS system (for example the
ionisation technique). A number of examples of the application of MS to on-line monitoring
can be found in the review by Fabris and this demonstrates how such considerations can be
overcome.[20]
Del Orco and colleagues continuously monitored a reaction by sampling a small volume of
the reaction liquor into a flow of carrier solution which was eventually transferred to the
bench-top mass spectrometer.[21] Zhu et al and McCullough et al independently
demonstrated the application of extractive electrospray ionisation (EESI) to monitor a batch
process.[22,23] In order to obtain greater specificity for real time reaction monitoring Harry et
al and Roscioli et al have independently demonstrated ion-mobility mass spectrometry (IM-
MS) for pharmaceutical process understanding.[24, 25] Chen and Lin have recently reviewed
the monitoring of chemical transformations by MS using a variety of ion sources for direct
analysis and focussing on the range of ambient ionisation techniques.[26]
Miniaturised, lightweight and portable atmospheric pressure ionisation (API) mass
spectrometers further facilitate on-line MS for process analysis.[27] which allows the system
to be positioned with any process under investigation. The potential of such devices has
been demonstrated in two recent publications. In the first, the device was coupled to a
Uniqsis Flow Syn continous flow chemistry reactor.[28] In the second Bristow and co-workers
monitored the progress of a continuous flow Hofmann rearrangement reaction.[29] In this
study reactent dilution was achieved by transfer of small aliquots of sample solution into a
sampling make-up flow, followed by electrospray ionisation. The setup was able to detect at
least seven compounds in the reaction mixture when used on-line. It was identified in this
study that optimisation of make-up flow composition and sample dilution factor is
important in understanding reaction optimisation. This is analogous to the observations on
mixing recently reported by Foley et al for on-line NMR experiments.[11]
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Demonstrating the broader applicability of on-line MS to monitor pharmaceutical processes,
Ray and co-workers reported the on-line analysis of a tablet dissolution process. [30]
Individual dissolution profiles were obtained in real time for a tablet containing three active
pharmaceutical ingredients and lactose. Such detailed information on release is important
for design and development of pharmaceutical formulations.
As described many individual techniques have been applied to process monitoring. They
have their own unique advantages, some limitations and therefore on-line combinations are
of interest. One potentially powerful option is the combination of NMR and a mass
spectrometer. The quantitative and qualitative power of NMR and the inherent sensitivity of
the MS, would provide an analytical system that would allow the typically wide
concentration range of the components of a chemical process to be studied in a single
experiment. This is technically challenging. However, the combination of LC-NMR-MS has
been demonstrated previously and illustrates the potential. In 2003 Corcoran and Spraul
published a review outlining the application of LC-NMR-MS in drug discovery. [31] The review
describes solutions to the challenge of coupling the three techniques and a range of
applications including synthesis of natural products, combinatorial chemistry and drug
metabolism and pharmokinetics (DMPK). There are a number of individual applications of
the approach described in the literature. Examples of specific pharamaceutical applications
have been described in separate publications by Shockor, Hansen and their co-workers. [32,
33] In the former report the detection and characterisation of xenobiotics and metabolites in
human urine is described. In the latter study, LC-NMR-MS was applied to the structural
characterisation of the constituents of an extract from a plant natural product.
In this study on-line NMR-MS to monitor a batch chemical process at typical process
concentrations up to 71.0 mg/mL has been investigated. In this experiment the multi-
component sample mixture is transferred continually to the NMR and MS for analysis. This
approach differs from LC-NMR-MS where stop-flow experiments and LC pre-separation
delivers the individual components to the NMR and MS. Also, sample preparation and
chromatographic separation results in concentrations of a more appropriate level for the
two techniques.
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For on-line NMR-MS, the optimisation of the instrumental combination to cover a broad
dynamic range of reactant and product concentration (for example 0.01 mg/mL – 62.7
mg/mL, typical of process concentrations) is described. Individually, the instruments are
unable to monitor this entire concentration range. NMR suffered poor signal to noise at the
lower level of concentrations investigated and MS suffered from non-linearity of response at
high sample concentrations due to MS signal (detector) saturation. Following this
optimisation study, the system was applied to monitor a batch chemical process and this is
also described.
Experimental
Materials: All materials, unless specified, were sourced from Sigma-Aldrich Company Ltd.
(Gillingham, United Kingdom) at the highest available purity. Ultrapure water (18.2 MΩ) was
obtained from a MilliQ unit (Watford, Hertfordshire). Metmercazole and Pyrmetazole, the
product of the reaction studied were available at AstraZeneca. All of these materials were
fully characterised by proton NMR and LC-MS prior to use. Pyrmethyl chloride hydrochloride
was also synthesised from pyrmethyl alcohol (from AstraZeneca) as required.
Equipment setup: An EasyMax 102 Advanced Synthesis Workstation (Mettler-Toledo Ltd.,
64 Boston Road, Beaumont Leys, Leicester, LE4 1AW) with a 100 mL reaction vessel was
used for all reactions and was coupled on-line with a Bruker Avance 500 MHz spectrometer
1
(Bruker Biospin, Rheinstetten, Germany). Both conventional H experiments and solvent
1
suppression H experiments were employed (Bruker noesy1D pre-set solvent suppression
pulse program noesygppr1d). PEEK tubing with a standard stainless steel HPLC mobile phase
inlet filter was inserted into the reaction vessel and was connected to an Ismatec (IDEX
Health & Science GmbH, Wertheim, Germany) IP 65 rotary piston pump. Stainless steel
HPLC tubing (0.5 mm ID) tubing was also fed from the pump out of the fume cupboard
housing the reactor. PEEK tubing was connected to the stainless steel tubing and attached
to the flow-cell of the NMR spectrometer. This contained a conventional 5 mm NMR tube
with a leak sensor.[15] PEEK tubing was attached to the exit port of the flow-cell and fed
through the heated transfer lines back into the reaction vessel completing a circular sample
path and has been described previously.[15] All tubing external to the reaction vessel and
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NMR magnet bore was insulated with high density polyethylene foam which ran alongside
the heated transfer lines from a Julabo oil bath to manintain temperature control. A
thermostatic heater/chiller was used to pump oil through tubing placed in parallel with the
reaction flow and inside the insulating foam. This provided temperature control from the
reaction vessel to the NMR.
To complete the on-line NMR-MS experimental configuration, a Waters Acquity QDa mass
spectrometer (Waters Limited, Elstree, England) was coupled into the flow path described
above. The QDa is a small footprint mass spectrometer (dimensions: 35.3 × 20 × 75cm) that
is transportable because of its low weight (approx. 30 kg) and dimensions. To transfer
samples to the mass spectrometer, the sample return flow was connected into a Rheodyne
100-000 MRA (IDEX Health & Science, Wertheim-Mondfeld, Germany). The MRA samples
small aliquots of solution from the process flow, providing a 1 in 1000 dilution (in our typical
set-up) and passes these into a sampling make-up flow (0.1 % formic acid (v/v) in 60:40 (v/v)
acetonitrile:water) which is required for positive ion electrospray ionisation. Reaction
mixture not sampled exits the MRA by a third port and is returned to the reaction vessel and
this has been described previously.[29, 30] The electrospray sampling make-up flow was
delivered to the MRA by a binary HPLC pump (Waters Limited, Elstree, England) at a typical
flow rate of 1 mL/min. The sample flow entering the MRA was fitted with a KrudKatcher
Classic 0.5 μm stainless steel filter (Phenomenex, Macclesfield, England) to prevent any
particulates from damaging/blocking the MRA.
Samples were analysed by positive ion electrospray ionisation and for all experiments the
mass spectrometer was operated in scan mode typically in the range 100-1000 m/z with a
scan time of 0.5 seconds. Other key MS parameters were source temperature of 100 °C, a
probe temperature of 300 °C, a capillary voltage of 1.5 kV and a cone voltage 25 V.
Nebuliser nitrogen gas flow rate was regulated at 6.75 ±0.25 bar (98 ±4 psi) outlet pressure
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Experimental (i) - The assessment of the MS and NMR linearity
Pyrmethyl chloride hydrochloride solutions were prepared at a range of concentrations
from 0.02% to 120% of the typical reaction concentration equivalent to 0.01 mg/mL and
62.7 mg/mL. Pyrmetazole solutions were prepared at a range of concentrations from 0.02%
to 100% of the reaction concentration equivalent to 0.02 mg/mL and 71.0 mg/mL. These
solutions (each calibration solution introduced separately and sequentially) were analysed
to assess a number of key experimental parameters for the combination of NMR and MS,
with solution concentrations confirmed by HPLC. The solutions were intended to mimic
reaction conditions allowing MS and NMR linearity to be assessed. The NMR-MS reaction
monitoring system was employed as described with individual solutions pumped through
the NMR flow-cell, into the active splitter (to provide a 1000:1 dilution for MS) and back into
the reaction vessel. The pump flow rate was set to 1.4 mL/min to prevent high back-
pressure resulting in NMR flow-cell leakage. A time delay of 8 minutes was observed from
the solution being provided to the pump, to a signal intensity plateau on the mass
spectrometer. For this reason, all sample solutions analysed in this experiment were
circulated for around 10 minutes before NMR acquisition and MS analysis. The time window
used for averaging the MS data was approximately 4 minutes post reaching maxima. In
these experiments all solutions were analysed from low concentration to high concentration
in order to minimise carryover and system contamination.
Experimental (ii) - On-line reaction monitoring
The nucleophilic substitution reaction to form pyrmetazole and monitored by NMR-MS is
shown in Scheme 1. Pyrmethyl chloride (5.39 g) was charged to the reactor and dissolved
in methanol (60 mL). The pump transferring reaction mixture from the reactor to the NMR
and MS was set to a flow-rate of 0.65 mL/min. A suspension of metmercazole (4.49 g) in
methanol (30 mL) was then added. The solution was heated to reflux and the jacket
temperature maintained at 70 ˚C with a condenser in place.
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The temperature in the NMR was set to 313K to match the temperature of the incoming
reaction liquor. It should be noted that the heated transfer lines from the reaction vessel
only insulate the stainless steel transfer tubing to the top of the NMR magnet. At this point
it is connected to an “un-insulated” section of PEEK tubing that runs down the upper barrel
of the magnet, resulting in an approximate temperature drop of 25K.
A sample containing the two starting materials and product was also analysed off-line by
NMR in non-deuterated methanol in order to identify suitable NMR signals for reaction
monitoring (Figure 1).
Results and Discussion
1. Assessment of the relative linearity of on-line NMR and MS using Pyrmethyl Chloride
Hydrochloride
Figure 2 shows the change in response for both NMR and MS on-line as a function of the
concentration of the pyrmethyl chloride hydrochloride solutions. This was in the range
0.02% to 120% of the reaction concentration, equivalent to 0.01 mg/mL and 62.7 mg/mL.
The NMR integrals recorded show a very strong linear correlation with solution
concentration (Figure 2a) however, the MS ion abundance shows a significant deviation
from linearity beyond 2% (1.25 mg/mL) which is attributed to MS signal (detector)
saturation (Figure 2b). This is more clearly illustrated in Figure 2c where MS ion abundance
is plotted as a function of concentration in the range 0.01% to 10% (6.0 mg/mL) of the
reaction concentration.
It was also important to evaluate the effect of other known reaction components on MS
response. Therefore, a second set of pyrmethyl chloride hydrochloride solutions were
prepared in the presence of pyrmetazole in the range 0.01% to 10% of the reaction
concentration. The pyrmetazole concentration was varied to qualitatively mimic the
reaction composition. The solution with 12 mg/mL pyrmetazole is out of this trend to ensure
that the linearity of the pyrmetazole concentration range wasn’t artificially creating the
apparent pyrmethyl chloride linearity. If this were the case this particular solution would
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have given a large signal in the residuals plot created with the MiniTab statistical analysis
(not shown).
Figure 3 shows extended linearity over a wider concentration range in the presence of
pyrmetazole, which is likely to result from ion suppression reducing the overall ion
formation from pyrmethyl chloride hydrochloride. For example, the MS ion abundance was
5.5 x 107 at 6 mg/mL in the absence of pyrmetazole (Figure 2c) and was 3.0 x 107 in the
presence of pyrmetazole (Figure 3). However, it is also important to note that the lowest
solution concentration was still detected maintaining the required MS sensitivity to study
this reaction system.
In Bruins key publication on the mechanisms of electrospray, it is concluded that although
electrospray is a quantitative ion source for mass spectrometry, the signal saturates at
around 10 μmol dm-3 due to the surface of the droplet emitting ions being full.[34] For
pyrmethyl chloride, a concentration of 10 μmol dm-3 corresponds to a reaction
concentration of 2.22 mg/mL prior to MRA dilution. This corresponds to around 4 % of the
maximum reaction concentration of pyrmethyl chloride. Therefore, the limited MS linearity
at the concentrations examined here is explainable.
One can see in Figure 3 that the mass spectrometer response begins to deviate from
linearity at concentrations greater than 3 mg/mL (5 % of the reaction maximum
concentration) compared to 1.25 mg/mL in the absence of the matrix. These data therefore
demonstrate that pyrmethyl chloride hydrochloride response at levels between 0.01 % and
5 % of the maximum reaction concentration show a linear response with concentration.
These experiments were also repeated and verified using pyrmetazole in the range of
concentrations 0.02% to 100% of the reaction concentration equivalent 0.02 and 71.0
mg/mL, with equivalent results for linearity for MS and NMR.
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2. Assessment of the relative LOD and LOQ for on-line NMR and MS using Pyrmethyl
Chloride Hydrochloride
To further illustrate the advantage of coupling the two techniques an assessment of
LOD/LOQ for on-line NMR-MS was carried out. Though it is clear that NMR has an advantage
over MS in terms of the linear dynamic range over this broad process concentration range,
MS out performs NMR in terms of overall sensitivity and it’s ability to detect low level
reaction components. Figure 4 shows a waterfall plot for the 1H NMR data of pyrmethyl
chloride hydrochloride at a range of concentrations (0.01 mg/mL to 6 mg/mL). A signal to
noise ratio of 17:1 was observed for the sample solution at 0.5 mg/mL and 2:1 for the
sample solution at 0.06 mg/mL (signal to noise values were determined after 8 scans with a
3s recycle time and were measured between 8.8 - 8.4 ppm, with 1 ppm of noise (10 ppm – 9
ppm)). Below this concentration the signal cannot be distinguished from noise. By contrast
the m/z 186 ion of pyrmethyl chloride hydrochloride was clearly detected at the lowest
concentration, illustrating on-line MS sensitivity of at least 5 times greater than NMR with
this experimental configuration. By MS, the strong correlation between low-level solution
concentration and signal intensity demonstrates that good S:N can be achieved at low
concentrations of pyrmethyl chloride hydrochloride.
Following this initial optimisation and development of the understanding of the capability,
the combined system was capable of monitoring the process reaction in the concentration
range 0.02% - 100%. However, as the MS linear dynamic range was limited to approximately
3 mg/mL (2%) at this stage (Figure 3), further optimisation of the MS experiment was
undertaken to improve this measurement capability.
3. Extending the linearity of on-line MS
To extend the linear dynamic range of the mass spectrometry experiment, a number of
parameters were further optimised whilst analysing pyrmethyl chloride hydrochloride
solutions (0.01% to 10% relative reaction concentrations) in the presence of of pyrmetazole.
The active splitter sample dilution parameter was further increased to 4000:1 and 6666:1
(from the initial 1000:1) using a sampling make-up flow of 2 mL/min. Such a high flow rate is
unsuitable for direct coupling to the electrospray ion source of the QDa and was therefore
combined with a post active splitter (pre-MS) flow split of approximately 1.65 : 0.35 (waste :
MS). This optimised system resulted in the extension of the MS linear dynamic range for
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pyrmethyl chloride hydrochloride to 6 mg/mL (10% relative reaction concentration) due to
this significantly increased dilution step (Figure 5), whilst at the same time maintaining the
ability to detect the lowest concentration solution (0.01% relative reaction concentration).
4. On-line NMR-MS Reaction Monitoring of a Nucleophillic Substitution Reaction.
Using the optimised NMR-MS reaction monitoring configuration, the nucleophilic
substitution reaction to produce pyrmetazole was studied (Scheme 1). Both NMR and MS
spectra generated online during reaction monitoring (Figure 6) were found to be equivalent
to standard spectra supporting peak assignment. Although the order of the NMR signals
around 7 ppm are slightly different when measured on-line, assignment could be made
based on line shape and multiplicity. These differences are likely to be due to concentration,
pH and temperature differences as the reference NMR spectra were acquired at 300 K,
whereas during on-line analysis the acquisition temperature was set to 313 K. The
temperature in the NMR was set to 313 K to match the temperature of the incoming
reaction liquor. It should noted that shimming at 313 K was not a major issue. The half-
height line width (for the 0.5 mg/mL solution) of the signal at 8.65ppm was 6.5Hz. Whilst
this is broader than would have been liked, the signal of interest is well resolved from all
other signals and the drawing of an accurate integral was straight forward.
On-line monitoring of the reaction by MS and NMR generated the reaction profiles shown in
Figure 7. The quantitative reaction profiles (Figure 7a) were produced by integrating the
peaks of interest δH 8.7 and 8.5 on waterfall plot (Figure 8). It is interesting to note here that
the reaction concentrations begin to plateau much sooner by MS than by NMR, however the
explanation for this is not clear at this time.
Conclusion
The on-line combination of NMR and MS has been shown to have clear advantages for
reaction monitoring over the individual techniques. Using a flow-tube, NMR can provide
structural and quantitative kinetic information for a reaction. MS has shown to be highly
advantageous for structure elucidation and has a clear sensitivity advantage when detecting
low level components and impurities in the reaction.
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In this study, linearity of NMR response for both pyrmethyl chloride hydrochloride and
pyrmetazole at concentrations between 0.02 % – 120 % and 0.02 % – 100 % respectively has
been demonstrated. Linearity of MS response was initially demonstrated to be limited for
pyrmethyl chloride hydrochloride to concentrations between 0.01 % and 2 % of maximum
process concentration. Further optimisation extended this range to 10 % or 6 mg/mL. The
limited linear dynamic range of MS at high concentrations and the poorer sensitivity on
NMR for low level components was overcome by combining the two techniques in tandem
on-line. Finally, the combination was demonstrated successfully by direct monitoring of a
nucleophilic substitution reaction.
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Scheme 1. The nucleophillic substitution reaction used to form pyrmetazole. Compound
names (left to right) are pyrmethyl chloride hydrochloride, metmercazole and pyrmetazole.
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Table 1. The solution concentrations used to determine the effect of additional reaction
components on the MS response for Pyrmethyl chloride hydrochloride.
Concentration of Pyrmethyl
chloride hydrochloride
Concentration of
pyrmetazole
(mg/mL)
m/z 186
abundance
(× 107 ) in the
absence of
m/z 186
abundance
(× 107 ) in the
presence of
(% of reaction maximum)
sample matrix sample matrix
0.01
0.05
0.2
0.5
1
12
24
21
18
15
12
9
0.240
0.703
1.13
2.87
3.36
4.63
4.99
5.83
0.0217
0.0281
0.122
0.251
0.588
1.20
2
5
2.33
10
6
3.43
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Figure 1. NMR signals selected for reaction monitoring.
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(a)
2500
2000
1500
1000
500
0
0
10
20
30
40
50
60
Solution Concentration / mg/mL
(b)
700
600
500
400
300
200
100
0
0
10
20
30
40
50
60
Solution Concentration / mg/mL
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(c)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
-1.00
0
1
2
3
4
5
6
Pyrmethyl chloride concentration / mg/mL
Figure 2. Evaluation of the linearity of the NMR and MS over the process concentration
range (a) NMR linearity plot for pyrmethyl chloride hydrochloride, (b) MS linearity plot for
pyrmethyl chloride hydrochloride and (c) MS low-level linearity assessment of pyrmethyl
chloride hydrochloride in the absence of pyrmetazole.
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4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
y = 0.594x + 0.1704
R² = 0.9631
0
1
2
3
4
5
6
Pyrmethyl chloride hydrochloride / mg/mL
Figure 3. MS low-level linearity assessment of pyrmethyl chloride hydrochloride in the
presence of varying levels of pyrmetazole.
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Figure 4. Waterfall plot of pyrmethyl chloride hydrochloride relevant 1H signal
corresponding to solution concentrations of 0.01, 0.03, 0.06, 0.5 and 6 mg/mL (1-5
respectively).
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Figure 5: Extension of MS linear dynamic range at process concentrations - plot of ion
abundance (107) as a function of the calibration standards (mg/mL) of pyrmethyl chloride
hydrochloride in the presence of sample matrix and with the addition of a pre-MS flow split.
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Metmercazole
Pyrmetazole
Pyrmethyl
chloride
hydrochloride
AB_140415_02 1064 (8.954)
Scan ES+
5.34e7
330
100
186
188
331
332
333
168
749
189
721
700
152
752
0
100
m/z
1000
200
300
400
500
600
800
900
Figure 6. 1H NMR and MS spectra acquired on-line during reaction monitoring.
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(a)
2450
1950
1450
950
Pyrmetazole
Pyrmethyl chloride
450
-50
0
10
20
30
40
50
60
Time / minutes
(b)
1.20E+08
1.00E+08
8.00E+07
6.00E+07
4.00E+07
2.00E+07
0.00E+00
Pyrmetazole
Pyrmethyl chloride
0
5
10
15
20
25
30
35
40
45
Time / minutes
Figure 7. Reaction profiles generated using (a) NMR integrals and (b) the ions at m/z 186
and 330 m/z corresponding to starting material and product respectfully for the
nucleophillic substitution reaction.
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Figure 8. NMR waterfall plot from the reaction monitoring acquisition (y axis = intensity and
z axis = acquisition number).
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