Monitoring reactions

The reactions that produce drug substance are relatively easy to automate. However, automation by itself does not guarantee a robust process. It is still necessary to rework, and validation requires consideration of a worst-case 

In this example, mid-infrared (mid-IR) could equally be used. There are many other examples of where a specific spectroscopy could be apphed to a specific monitoring problem, in addition to NIR and mid-IR, there are examples using UV, Raman and fluorescence. However it is very rare that a specific question is posed. In the majority of cases, the abihty to monitor one step in what could be a multi-step reaction, does not assure reUable performance of the entire process. 

This increased spectral information can be maximised still further by applying the same chemometric principles used in NIR. If a derivative is apphed to the data, we can resolve overlapping absorptions to differentiate between components with essentially similar UV spectra. This does not however address fundamental issues with probe design. UV spectroscopy, like NIR uses transmission probes 

Typical UV spectra Fig. 9.6 Increased information with increased UV spectral resolution. 

These spectra were obtained through the oil-filled heating jacket of a laboratory scale automated reactor. Although undoubtedly a useful tool, this head is not a solution to general reaction monitoring. It copes well with the presence of bubbles and is excellent for monitoring slurries during crystallisation, but if the reaction mixture is black, as is often the case in the presence of catalyst, no reflectance is measured. 

In a third example, a reaction common in the pharmaceutical industry, reductive amination was used, such as for the production of secondary amines. In addition, this reaction is a challenge to monitor in that the imine intermediates are often susceptible to hydrolysis, making off-line analytical methods such as HPLC difficult. 

FIGURE 15.9 Example of reductive amination reaction monitored in real time. The reaction was monitored continuously for a period of 300 s. The changes in these spectra occur in the first 50 s. (From Roscioli et al., Real time pharmaceutical reaction monitoring by ion mobility-mass spectrometry, Ana/. Chem. 2012. With permission.) 

Depending on the rate of the enzyme reaction, the reaction mixture can be sampled for mass spectrometry measurements in one of the following three ways [1,2]. 

Off-Line Reaction Monitoring For slow enzyme reactions and long-lived intermediates, off-line reaction monitoring is more convenient. In these methods, the known amounts of an enzyme and a substrate are mixed together and incubated at physiological conditions. Aliquots are withdrawn at predetermined intervals, and the reaction is quenched immediately. The time course of the reaction can be monitored with mass spectrometric analysis immediately or later, at a more convenient time, by using either fast atom bombardment (FAB) [7], continuous-flow (CF)-FAB [7], matrix-assisted laser desorption/ionization (MALDI) [8], or electrospray ionization (ESI) [9-11]. Established quantitation procedures can be employed to monitor the concentration of the reactant or product (usually, the latter) (see Chapter 14). As an example, an appropriate internal standard that has no affinity for the enzyme can be added to the reaction mixture to improve 

At present, ESI is an ideal choice for continuous onUne monitoring of enzyme reactions [13-15]. The electrospray syringe itself can be converted into a reaction 

Rapid-Mixing or Stop-Flow Monitoring To elucidate the mechanism of very fast enzyme reactions, it is critical to monitor the initial events before the system has a chance to attain a steady state. In many cases, the intermediate transient species exist for only few milliseconds. The fast enzyme reactions can be monitored readily by combining rapid mixing and quenching devices with mass spectrometry. The multiplex detection and fast scanning capabilities of time-of-flight (TOF) and Fourier transform (FT)-ion cyclotron resonance (ICR) mass spectrometers make them ideal for this combination. 

Most people who are new recruits to the research labs learned their basic skills for carrying out reactions in an undergraduate laboratory. Inevitably, most of the organic chemistry undertaken in these lab classes involves following recipes, which have been well tried and tested. Therefore the conditions and the time taken for the reactions to reach completion are well 

In deacylation, as the enzyme cleaved the phenylacyl group, phenylacetic acid was formed, which lowered the pH of the reaction medium. Base was added to maintain the starting pH. (Note Use of ammonium hydroxide led to the formation of desilylated byproducts desilylation was eliminated when bicarbonates were used.) This approach was not required in the acylation reaction. At pH above 7.5 the (R)-and (S)-amines are practically insoluble in water. Organic solvents were used to extract the free amines from the aqueous reaction medium at pH 8.0. p-Fluoro-benzoyl, 1-naphthoyl, and phenylacetyl derivatives of the racemic amine were prepared and their behavior on the chiral HPLC column was studied. Based on ease of preparation and HPLC analysis, the 1-naphthoyl derivatives (Fig. 7) were preferred. Reversed phase HPLC analysis on a Vydac-C18 analytical column used a gradient of acetonitrile (0.1% triethylamine) in water (0.05% phosphoric acid) to quantify the total amide in the reaction mixture. Chiral HPLC analysis on (S,S) Whelk-O Chiral column used isopropanol hexane (30 70) as a solvent system to separate and quantify the (R)- and (S)-enantiomers. 

This example illustrates several important lessons. First, this application takes advantage of Raman signal enhancements to measure concentrations normally considered too low for Raman spectroscopy. Extremely strong Raman signal is a hallmark of carotenoid measurements. Traditionally, this enhancement has been considered a resonance effect but others believe it is due to coupling between 7r-electrons and phonons.37 It can be beneficial to use such enhancements whenever possible. 

Second, the results of off-line experiments do not necessarily predict the success or failure of on-line experiments. Off-line experiments had previously produced poor results 

it can be difficult to match the results from samples removed for off-line analysis (reference values) with the right spectra. If the system is changing rapidly, removed samples may be quenched incompletely and continue to react. This will introduce bias into reference values. Careful procedures must be developed to minimize bias between on-line and off-line results. 

Fourth, while fluorescence was not a problem in this system, some bioreactor components may be highly fluorescent. Until more experience is gained, this makes it difficult to make generalizations about the likelihood of success with new systems. Finally, water is a weak Raman scatterer and can be hard to track. While Ulber etal. considered the challenge in obtaining a spectrum of the aqueous cell culture broth to be a disadvantage, others consider it an advantage since these solutions overwhelm mid- or near-infrared detectors.33 34 36 

Lee etal. determined the concentration of glucose, acetate, formate, lactate, and phenylalanine simultaneously in two E. coli bioreactions using Raman spectroscopy.33 The bioreactor was modified to have a viewing port, and spectra were collected through the port window. This enabled researchers to sterilize the reactor and ensure that it would never be contaminated by contact with the instrument. 

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SRM. selected reaction monitoring SSMS. spark source mass spectrometry... 

The most common modes of operation for ms/ms systems include daughter scan, parent ion scan, neutral loss scan, and selected reaction monitoring. The mode chosen depends on the information required. Stmctural identification is generally obtained using daughter or parent ion scan. The mass analyzers commonly used in tandem systems include quadmpole, magnetic-sector, electric-sector, time-of-flight, and ion cyclotron resonance. Some instmments add a third analyzer such as the triple quadmpole ms (27). 

In this context it is important to note that the detection of this land of alkali cation impurity in ionic liquids is not easy with traditional methods for reaction monitoring in ionic liquid synthesis (such as conventional NMR spectroscopy). More specialized procedures are required to quantify the amount of alkali ions in the ionic liquid or the quantitative ratio of organic cation to anion. Quantitative ion chromatography is probably the most powerful tool for this kind of quality analysis. 

Essential prerequisites for the evolution of combinatorial methods were the progress in reaction monitoring and analytics. Of specific importance was the analytics... 

The TBDMS ether was dissolved in MeCN containing 5-30% of aqueous HF (40%), and the course of the reaction monitored by direct t.l.c. analysis. When deprotection was complete, chloroform and water were added. Normal isolation procedures then gave the free alcohol. 

A mixture of the a/J-unsaturated ester (14mmol), t-butyldimethylsilane (18 mmol) and tris(triphenylphosphine)rhodium(i) chloride (0.56 mmol) was placed in a pre-heated (100°C) oil bath, and the course of reaction monitored by i.r. spectroscopy. On completion (ca. 30 min) the product was isolated by direct distillation (60-88%). 

The kinetics of resole cure reactions monitored via FTIR suggest that a diffusion mechanism dominates below 140°C. The cure above 140°C exhibits a homogeneous first-order reaction rate. The activation energy of the cure reaction was -"-49.6 kJ/mole.66... 

Abstract Current microwave-assisted protocols for reaction on solid-phase and soluble supports are critically reviewed. The compatibility of commercially available polymer supports with the relatively harsh conditions of microwave heating and the possibilities for reaction monitoring are discussed. Instrmnentation available for microwave-assisted solid-phase chemistry is presented. This review also summarizes the recent applications of controlled microwave heating to sohd-phase and SPOT-chemistry, as well as to synthesis on soluble polymers, fluorous phases and functional ionic liquid supports. The presented examples indicate that the combination of microwave dielectric heating with solid- or soluble-polymer supported chemistry techniques provides significant enhancements both at the level of reaction rate and ease of purification compared to conventional procedures. 

Although solid-phase synthesis revolutionized synthetic organic chemistry and triggered the development of combinatorial chemistry, it still exhibits several shortcomings originating from the nature of heterogeneous conditions, such as lower reaction rates and difficulties in reaction monitoring. 

The copper atom-acetylene matrix-reaction, monitored originally by esr spectroscopy (60) has now been investigated by IR/UV-visible spectroscopy (144), and there is general agreement on the identification of two mononuclear species, CuCCaHali.. The esr/IR/UV-visible... 

The MS-MS equivalent of this technique is known as selected-decomposition monitoring (SDM) or selected-reaction monitoring (SRM), in which the fragmentation of a selected precursor ion to a selected product ion is monitored. This is carried out by setting each of the stages of mass spectrometry to transmit a single ion, i.e. the precursor ion by MSi and the product ion by MS3 (see Figure 3.8 above). 

This practical set-up may also be used for reaction monitoring by placing the capillary into a reaction mixture and continually acquiring mass spectra, which thus allows the analyst to examine changes in its composition. 

Table 5.2 Selected-reaction monitoring (SRM) transitions nsed for MS-MS detection of the pesticides studied in the systematic investigations on APCI-MS signal response dependence on eluent flow rate. Reprinted from J. Chro-matogr.. A, 937, Asperger, A., Efer, J., Koal, T. and Enge-wald, W., On the signal response of various pesticides in electrospray and atmospheric pressure chemical ionization depending on the flow rate of eluent applied in liquid chromatography-mass spectrometry , 65-72, Copyright (2001), with permission from Elsevier Science...

Figure 5.58 Reconstructed LC-MS-MS ion chromatograms for selected-reaction monitoring of methoxyfenozide using the m/z 367 to m/z 149 transition from the continual post-column infusion of a standard solution of analyte during the HPLC analysis of a...

Figure 5.64 LC-UV and LC-MS-MS (multiple-reaction monitoring (MRM)) traces from the analysis of a synthetic mixture of four native and five oxidized deoxynucleosides (for nomenclature, see text). Reprinted by permission of Elsevier Science from Comparison of negative- and positive-ion electrospray tandem mass spectrometry for the liquid chromalography-landem mass speclrometry analysis of oxidized deoxynucleosides , by Hua, Y., Wainhaus, S. B., Yang, Y., Shen, L., Xiong, Y., Xu, X., Zhang, F., Bolton, J. L. and van Breemen, R. B., Journal of the American Society for Mass Spectrometry, Vol. 12, pp. 80-87, Copyrighl 2000 by Ihe American Society for Mass Spectrometry.

Figure 5.66 Molecular structures of Idoxifene and its deutrated internal standard ds-Idoxifene. Reprinted from J. Chromatogr., B, 757, Comparison between liqnid chromatography-time-of-flightmass spectrometry and selected-reaction monitoring liqnid chromatography-mass spectrometry for qnantitative determination of Idoxifene in hnman plasma , Zhang, H. and Henion, I., 151-159, Copyright (2001), with permission from Elsevier Science.

Figure 5.69 Calibration curves obtained from (a) LC-ToF-MS and (b) LC-MS-MS using selected-reaction monitoring for Idoxifene in human plasma, fortified from 5 to 2000 ngml for LC-ToF-MS and 0.5 to 1000 ngml for LC-MS-MS with a triple quadrupole is the correlation coefficient, a measure of the quality of calibration (see...

Selected-reaction monitoring (See Selected-decomposition monitoring)... 

Molecules are too small and much too numerous to follow on an individual basis. Therefore, a chemist interested in measuring the rate of a reaction monitors the concentration of a particular compound as a function of time. The concentrations of reactants, products, or both may be monitored. For example, Figure 15-6 shows some experimental data obtained from a series of concentration measurements on the decomposition of NO2 ... 

Tian, Q. et al., Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring, J. Chromatogr. A, 1091, 72, 2005. 

Figure 2. Plot of rac-1 conversion versus time for colloidal and molecular catalytic systems, (a) Reactions morritored during 24 h for colloidal and molecular (Pd/I = 1/500, 1/2,000 and 1/10,000) catalysts, (b) Reactions monitored during one week for colloidal and molecular (Pd/I = 1/10,000) catalysts. (Reprinted from Ref. [44], 2004, with permission from American Chemical Society.)...

The method for chloroacetanilide soil metabolites in water determines concentrations of ethanesulfonic acid (ESA) and oxanilic acid (OXA) metabolites of alachlor, acetochlor, and metolachlor in surface water and groundwater samples by direct aqueous injection LC/MS/MS. After injection, compounds are separated by reversed-phase HPLC and introduced into the mass spectrometer with a TurboIonSpray atmospheric pressure ionization (API) interface. Using direct aqueous injection without prior SPE and/or concentration minimizes losses and greatly simplifies the analytical procedure. Standard addition experiments can be used to check for matrix effects. With multiple-reaction monitoring in the negative electrospray ionization mode, LC/MS/MS provides superior specificity and sensitivity compared with conventional liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/ultraviolet detection (LC/UV), and the need for a confirmatory method is eliminated. In summary,... 

LC/MS/MS. LC/MS/MS is used for separation and quantitation of the metabolites. Using multiple reaction monitoring (MRM) in the negative ion electrospray ionization (ESI) mode, LC/MS/MS gives superior specificity and sensitivity to conventional liquid chromatography/mass spectrometry (LC/MS) techniques. The improved specificity eliminates interferences typically found in LC/MS or liquid chro-matography/ultraviolet (LC/UV) analyses. Data acquisition is accomplished with a data system that provides complete instmment control of the mass spectrometer. 

Electrospray (Turbo lonSpray), negative ion mode MS/MS with multiple reaction monitoring (MRM) -4500 V... 

Figure 1 Selected reaction monitoring of the two primary chlormequat ions using MS/MS...

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