Chromatographic modeling using DryLab®4 software from Molnár-Institute saves time and resources across all three stages of the lifecycle of an analytical method. Method run time can easily be reduced 40-fold with improved selectivity when redesigning an old method, simultaneously reducing the development time by a factor of 12. US Pharmacopeia (USP) methods that frequently fail can be fixed in accordance with allowed changes (USP 621) using DryLab4. Chromatographic modeling can safeguard method robustness across the lifecycle for compendial methods, method development, and method transfers.

The lifecycle of analytical methods has three stages: Design, Qualification, and Continued Verification. Ideally, a design space is defined during the early stages of the method development. Methods design is a range of parameters within which the method performs as intended and within which the method is fit for use. ICH Q8(R2) states that “working within the design space is not considered as a change.” In addition, ICH Q12 (draft) introduces a post-approval management plan to be initiated at submission (Post-Approval Change Management Protocol or PACMP). It integrates flexibility and lowers the need for new validation when changes are made to the method in Stage 3 (i.e., continued verification).

When one runs into problems with a method in Stage 3, it may be necessary to return to design and development (Stage 1) to resolve the problems. This task can be an arduous one, especially when production is ongoing. Indeed, one department typically handles both Stage 1 method development and design, while the Stage 3 routine method use is handled by a different department, with tight timelines for production of results.

DryLab4 software from Molnár Institute can help with this challenge because it can optimize the performance of an existing method in three simple steps: input runs, peak tracking and create the model, which helps find the most robust separation within the existing design space. The input runs bracket the existing method conditions about the allowed variations for the multiple variables of the method. Peak tracking is the identification of peaks in the input runs. DryLab then creates the model.

Using just a few experimental runs, DryLab visualizes the chromatographic process interactively, running hundreds of simulations in a few moments to find the best separation conditions, highest critical resolution, shortest run time and the highest robustness. Furthermore, evaluating method robustness during method development, while defining the analytical target profile (ATP), ensures a working design space for post-approval management and flexibility in dealing with out of specification (OoS) results in routine work. DryLab can be used at all stages, as shown in Figure 1/Slide 6.

Compendial methods are Stage 3 methods by definition and do not require further method development and validation. However, in some cases, the method may need to be modernized. Such was the case with ebastine from a description in the European Pharmacopeia. The method was not fit for purpose because it was too long. The analytical work could not keep up with production. DryLab was used to reduce the analysis time from 160 minutes to less than 3 minutes in less than two weeks.

Based on prior knowledge and experience with similar projects, the parameters: gradient time tG (3-9 min), pH (2-3), temperature T (15-45°C), and ternary composition of the eluent B were considered the most probable potentially critical separation parameters for the method. DryLab also produced the Knowledge Management Document, complete with the original chromatograms, peak tracking, resolution maps, robustness testing, and confirmation of predictions. This improved method was accepted in five countries and is described in literature.

In another case, the routine method for amlodipine in the European Pharmacopeia was found to be not fit for purpose (Slide 18). Not only was it a long 45–60-minute isocratic method, but it also had coelutions and peak turnovers. Again, using only 12 experimental runs, and calculation of the model as shown in Fig.2, it became clear, that the separation in the middle group of 3 peaks was not based on pH-changes, but much more based on temperature fluctuations and had to do also with the gradient time selection.

The model enabled researchers to evaluate the resolution and robustness across the range of critical separation parameters. US Pharmacopeia <621> states, “multiple adjustments can have a cumulative effect on the performance of the system and are to be considered carefully before implementation.” Using DryLab to troubleshoot and understand the method does this using a virtual model that combines the multiple parameters in a 3D cube.

In Figure 2, the cross-hatch is set to the optimum area. However, when the user moves the cross-hatch around different areas of the cube, the chromatogram shows the predicted result as a chromatogram. The red area indicates areas of robustness. The gradient of color strips from orange to deep blue show how the critical resolution as the most important method parameter most likely to result in method failure and OoS results. Working near the boundary of the red zone results in poor method robustness. The critical pair for resolution is shown in red. As method parameters change, the critical peak pair may change also, which is reflected in the virtual chromatogram in the model. DryLab makes it very straightforward to take advantage of modern ultrahigh performance liquid chromatography equipment and to optimize a robust method with a less than six-minute run time.

Building robustness into the method as early as possible in method development will help safeguard against later OoS results. A method is robust if it remains unaffected by small but deliberate variations in method parameters. A robust method provides reliability when used in normal daily work with multiple operators, multiple instruments, and slight changes in columns from batch to batch. This was not often done in the past, perhaps because of the complexity of designing an experimental matrix that takes into consideration the cumulative effect of multiple factors. If the intended purpose of the method is quantification, then the first requirement for robustness is good resolution. The value of the critical resolution should be at least 1.5 ( = baseline resolution). A critical resolution of 2 for the critical pair is even better. Good resolution means one has a baseline separated chromatogram, which means one can more reliably integrate and quantify the peaks.

Pramipexole is a drug substance used to treat Parkinson’s disease or restless leg syndrome. The separation of pramipexole from its known impurities is described in the European Pharmacopeia as well as in the US Pharmacopeia. The method calls for a C18 stationary phase; recommended columns are Inertsil or Symmetry. The mobile phase is a pH 3 phosphate buffer with an ion pair reagent. The organic eluent is acetonitrile and the gradient range is from 20–40%B in 15 minutes at a temperature of 40°C. This method was used in the laboratory for a long time, but reliability has since declined, as shown in Figure 3/Slide 22.

Thus, researchers wanted to adjust the method to improve the robustness and to comply again with the system suitability testing. DryLab was used to create a model about the design space for the conditions observed in the official method to see if they were still at the optimum working point. Researchers ran 12 experiments across the range of the official method with the combinations of gradient times of 8 minutes, 16 minutes, and 24 minutes; pH values of slightly higher than 3.3, and slightly lower than 3; and temperatures of 27 °C, 40 °C, and 54 °C. Data were then imported into DryLab, which helped with peak tracking. DryLab then calculated the model and used the input data to run hundreds of virtual experiments to create the model and generate the resolution map (see Figure 4/Slide 27).

In summary, DryLab reliably models analytical methods, with good agreement between the predicted and experimental chromatograms. The Knowledge Management Document supported the continued pramipexol analysis using the redesigned robust method. Furthermore, researchers were able to modernize methods for use with the latest technology, both instruments and columns, as in another case with amlodipine. The use of Visual Chromatography Modeling is well received, with the ebastine method accepted at regulatory agencies in five countries. Finally, DryLab enables production labora-tories to readily fix methods at the end of their lifecycle to eliminate OoS results and improve productivity.

One promising research avenue is oxytocin for Alzheimer’s disease. In July, scientists in Japan reported a breakthrough in vitro study. Using a mouse model, they found that oxytocin could reverse some of the nerve cell damage that occurs in the early stages of the disease.

According to a leading theory about Alzheimer’s, the disease occurs when a toxic protein called beta-amyloid begins to clump around neurons in the brain. The neurons degenerate, which leads to a decrease in synaptic plasticity and ultimately to cognitive decline. A mechanism called long-term potentiation (LTP), which allows nerve cells to encode memories, is blocked.  

The researchers, from Tokyo University of Science and Kitasato University, took slices of mouse brain on a dish, and perfused the cells with beta amyloid to confirm that LTP inhibition did occur. Next, they perfused the cells with oxytocin and found that the damage was reversed. They also performed a follow-up experiment to show that oxytocin was indeed the chemical responsible. 

“Although oxytocin has been reported to participate in regulating learning and memory performance, no [previous] report has clarified its effects on the beta amyloid-induced impairment of synaptic plasticity,” says lead study author Professor Akiyoshi Saitoh, from Tokyo University of Science. 

“We found that oxytocin could reverse the effects of beta amyloid on mice hippocampal LTP in an in vitro study. Now, we have examined the effects in an in vivo study. In the near future we will be able to publish our behavioural data.” 

While much more research is needed – beginning with a proof of concept in animal models – Saitoh is hopeful that oxytocin could be used as an Alzheimer’s treatment further down the line. 

“We suggest the interesting possibility that oxytocin should be a completely novel therapeutic modality for the treatment of memory loss associated with cognitive disorders,” he says. 

Oxytocin has also been indicated as a treatment for the social difficulties associated with autism spectrum disorders (ASD). For both autistic and non-autistic people, higher levels of oxytocin appear to correlate with better performance in social tasks. It seems plausible that administering artificial oxytocin would help people who struggle in this area. 

As has been the case with a lot of oxytocin research, however, the studies have shown mixed results. On the one hand, oxytocin sprays have been shown to improve some aspects of social functioning in ASD, including emotion recognition and eye contact. 

On the other, these studies tended to involve a one-off dose of oxytocin, rather than long-term daily treatments. Studies involving longer-term treatments have not been conclusive. 

That said, there have been some promising findings, and additional clinical trials are underway. Researchers are also trying to find out how the hormone operates within the brains of those with ASD, as per a 2019 study that analysed post-mortem brain tissue. This study measured oxytocin receptors in certain regions of the brain and found differences among those with autism. 

There is also a promising strand of research that focuses on oxytocin as an adjunct to psychotherapy. The idea is that the oxytocin might increase the bond between client and therapist, and boost the effectiveness of the treatment. 

German psychiatrist Rene Hurlemann, along with Simone Shamay-Tsoory from Haifa University, are trialling oxytocin plus group psychotherapy for loneliness. In their previous research, they found that lonely people had the same baseline oxytocin levels as controls. This changed when subjects were asked to think about a happy memory – controls saw a rise in oxytocin, but lonely people did not. 

Moving beyond psychiatric and neurological conditions, oxytocin might one day be used treat osteoporosis. In a recent study on rats, ageing female rats were given oxytocin. Unlike the control group, they did not lose any bone density, possibly because the hormone mediates the action of oestrogen in bones.

What seems to be clear across the board is that different people respond to oxytocin in different ways, and we need to learn more about dosing, mechanism of action and a host of other variables. To give one particularly stark example – in a recent study, cocaine-addicted men benefited from oxytocin therapy but cocaine-addicted women did not. 

“Although the role of OXT [oxytocin] in the modulation of social behavior is today indisputable, the field of OXT-based pharmacotherapy is filled with many small trials that report incongruent results,” wrote the authors of a recent mini-review article, published in Frontiers in Psychiatry. “Efforts should be made to understand its neurobiological mechanism of action and effect based on individual variability in underlying neuropeptide biology.”

In short, while oxytocin has clear therapeutic potential, there are key questions that remain unanswered. And until we understand precisely how it works in the body, we may struggle to repurpose it as a drug.