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Abstract

Many compound collections are stored under the same temperature conditions, which can limit flexibility by increasing the processing time required for high-demand compounds. In this study, the authors wanted to evaluate the impact of a hybrid-storage approach where high-demand compounds are stored for a shortened time period at room temperature to expedite processing operations. The use of a Covaris adaptive-focused acoustics platform was also characterized as a potential enhancement or alternative to storage at elevated temperatures. This study evaluated the impact of temperature, exposure, and solubilization on overall compound quality for short-term storage. A small library of 25 representative compounds was evaluated over an 18-week period to monitor the change in purity and concentration by high-performance liquid chromatography with ultraviolet detection. The authors concluded that temperature had a significant impact on compound concentration, and the effects due to exposure cycles were minimal. A storage time of 12 weeks at room temperature resulted in minimal compound loss, but storage times beyond this would be unacceptable because of a >20% decrease in concentration. Finally, the acoustic solubilization protocol also increased the number of compounds at the target concentration with no impact on overall purity, leading to a potential for increased storage times at frozen temperatures.

Keywords

compound management storage conditions compound quality DMSO HPLC-UV quantitation acoustic solubilization

Introduction

Multiple groups have recognized that maintaining compound quality is dependent on both storage conditions and sample handling methods.^1–3 Our workflows within Amgen have been built around low-volume, single-use operational tubes that are periodically prepared as replicates from high-volume, multiuse archive tubes, both stored at temperatures of −8 or −20 °C. This design minimizes the number of times an archive tube must be accessed by instead relying on the single-use tubes for normal operations. This approach effectively serves the majority of the collection, but we have recently noticed that certain compounds enter stages of high demand for a shortened period (<3 months) of time. These high-demand compounds often require frequent replenishment from multiuse archive tubes, which can affect the number of freeze-thaw cycles a compound undergoes. The extended sample handling time can lead to a decrease in overall order fulfillment efficiency and degradation in sample quality from unnecessary exposure outside of the store. Although one potential solution might be to prepare more initial single-use tubes, it is often hard to predict how much material will be required, and the preferred approach has been to replenish on-demand.

One alternative approach is short-term storage at room temperature, which would eliminate the need for thawing and reduce sample handling time. Other groups have shown that compound quality can remain stable for 4 to 6 months^4–6 at room temperature, but the labware was significantly different from our archive operations, leading to questions around the form factor used to store compounds. In this study, we wanted to understand the impact of short-term storage at room temperature as compared to the effect of multiple freeze-thaw cycles on overall compound quality in labware relevant to our operations. For our characterization of freeze-thaw events, we were primarily interested in the cumulative effect of both the freeze-thaw and the water uptake that is common with sample handling during formatting operations. Investigations to date that have explored the impact of freeze-thaw cycles have not attempted to characterize these effects independently.^4,7,8 We believe it is more precise to discuss these combined effects as exposure cycles in place of the term freeze-thaw cycle, which may refer to only the freezing-thawing effects in the absence of sample handling.

We also wanted to investigate the possibility of implementing a sonication-based protocol to accelerate sample thawing and solubilization of compounds prior to analysis. Approaches using traditional sonication⁹ and recently introduced adaptive focused acoustics (AFA)¹⁰ have both been reported as possible methods to initially solubilize compounds in library wells. The AFA approach has been shown to be more amenable to compound management operations due to the increased sample throughput resulting from the use of higher energy acoustic waves.

In this report, we explored effects of temperature and exposure on compound concentration to understand the feasibility of short-term storage on high-demand compounds in our labware, storage volumes, and temperature stores. Previous studies have typically characterized the stability of compounds in a low-volume (<40 µL) 96- or 384-well plate format, which differ significantly from the high-volume (1.4-mL) Matrix tubes that Amgen has standardized for archive compound storage.^4,6–8 As we will demonstrate, the labware format can affect the experimental parameters, so it is important to consider the storage vessel form factor in any stability validation study. We also wanted to characterize the effectiveness of a resolubilization protocol using a Covaris AFA platform. This technology has received some attention for one-off compound solubilization,¹⁰ but we instead have focused on understanding the impact of multiple solubilization cycles over the life of a compound as might be typical in a compound management environment.

Materials and Methods

Study design

A total of 25 compounds were selected based on historical observations of samples that had previously shown solubility problems in DMSO stock after visual inspection of library compounds in solution. These compounds were further refined to maximize diversity based on cLogP (2–8) and MW (200–600), representative of typical samples in the collection. All compounds in the study contained at least one nitrogen atom and were a mix of acids, bases, and neutral compounds. Although these compounds represented the potentially “worst case” with respect to long-term solubility, this also ensured that we would be working with compounds that were known to be of a particular concern in a compound management setting. The study was divided into three separate data sets to independently characterize the impact of temperature, exposure, and solubilization on the concentration of compounds using different storage conditions and protocols. Each of the data sets used the same time points at 0, 2, 4, 6, 12, and 18 weeks for a total of six measurements over the course of the study, but the handling protocol was unique for each data set, and each compound was analyzed a single time (n = 1). The three studies, analysis time points, and protocols are summarized in Table 1 .

Table 1.

Experimental Design for Three Study Sets

Temperature Stability Set (Single-Use Tubes)	Temperature (°C)	t₀ (Initial)	t₁ (2 Weeks)	t₂ (4 Weeks)	t₃ (6 Weeks)	t₄ (12 Weeks)	t₅ (18 Weeks)
RT	25	X	X	X	X	X	X
Frozen	−8	X	X	X	X	X	X
Exposure Set (multiuse tubes)	Temperature (°C)	t₀	t₁	t₂	t₃	t₄	t₅
RT (cumulative freeze-thaw cycles)	25	O (0)	O (1)	O (2)	O (3)	O (4)	O (5)
Frozen	−8	O (0)	O (1)	O (2)	O (3)	O (4)	O (5)
Solubilization Set (multiuse tubes)	Temperature (°C)	t₀	t₁	t₂	t₃	t₄	t₅
Solubilization, RT (cumulative cycles)	25	O (0)	O (1)	O (2)	O (3)	O (4)	O (5)
Solubilization, frozen	−8	O (0)	O (1)	O (2)	O (3)	O (4)	O (5)

X = single-use tube analyzed at time point and discarded. O = multiuse tubes analyzed and returned for subsequent time points. RT, room temperature.

The temperature set samples were prepared as single-use tubes for each time point. This ensured that effects due to sample handling were minimized and any change in the sample concentration could be related to the storage temperature. The exposure and solubilization sets were prepared as multiuse samples such that the same tube could be used throughout the study. These were samples prepared from a single-source tube that was analyzed and then returned to the store for the subsequent time points. This resulted in the samples for the exposure and solubilization sets undergoing multiple exposure cycles versus a single cycle for the temperature set. In total, the exposure and solubilization sets underwent five exposure cycles over the course of the study.

Sample preparation, storage, and exposure

Stock concentrations were prepared in dry DMSO (<0.2 wt% H₂O by Karl-Fischer titration) to 10 mM from solid material. The stock solutions were aliquoted into an array of 1.4-mL Matrix storage tubes (BC3006; Thermo Scientific) using the volume described in the study design. The temperature single-use set consisted of 6 × 30-µL tubes, one for each time point, whereas the exposure and solubilization multiuse sets both consisted of a single 150-µL tube that would be analyzed multiple times. Once dispensed, the Matrix tubes were capped and put in the specified storage conditions. Room temperature (RT) samples were stored in a TTP comPOUND (TTP LabTech, Inc., Cambridge, MA) store at ambient temperature and humidity. Frozen samples were stored in a Remp store at −8 °C and 9% relative humidity (RH). Samples were removed only at the specified time point and were either discarded or returned to the store based on the study design described above. An additional control sample was prepared for the exposure set that went through all of the handling steps over the course of the study but was analyzed only at the final time point to account for any differences that may have occurred due to variable volumes being investigated. A control sample was prepared at the same concentration but at a volume of 600 µL (fourfold increase over exposure set) to act as a control against surface-to-volume effects for water uptake. All tubes were prepared as a single instance (n = 1) for this study.

Prior to analysis, samples for the given time point were removed from the store and allowed to equilibrate at ambient temperature for 30 min without mixing, followed by centrifugation for 2 min at 1000 rpm on an Allegra 6 (Beckman Coulter, Brea, CA) Centrifuge. Samples remained capped during this equilibration, with the caps only being removed prior to transfer into the analysis plate. An Evo 3000 (Tecan, Männedorf, Switzerland) protocol was used to dispense 15 µL of each sample into a 96-well polypropylene round bottom plate (Costar 3365; Corning, Corning, NY) followed by vacuum heat seal with a PlateLoc (Agilent Technologies, Santa Clara, CA) plate sealer. The dispensing protocol took approximately 15 min, after which tubes were recapped and returned to the specified temperature store, simulating real-world handling protocols.

Sample solubilization protocol

Each sample from the solubilization set of the study was subjected to a further protocol of 15 s at 200 V and 18 °C on a Covaris E210 Focused Acoustic Solubilzer (Covaris, Woburn, MA). This protocol was applied after sample thawing but before samples were uncapped to limit water uptake concerns. The entire protocol took under 30 min to complete for a 96-tube rack, further minimizing variables introduced by sample handling. Samples were immediately dispensed in the 96-well analysis plate after completion of the Covaris solubilization protocol.

Sample analysis by LC-UV-MS

An Agilent 1100 binary high-performance liquid chromatography (HPLC) system connected to an MSD SL quadropole mass spectrometer (MS) with an electrospray ionization (ESI) source was used for all measurements. A Phenomenex (Torrance, CA) Gemini C₁₈ 2.0 × 50-mm column with 3-µm particles was operated at 1 mL/min and 50 °C using 100% water and 100% acetonitrile as mobile phases A and B, respectively, with 0.04% trifluoroacetic acid (TFA) added to each phase. The gradient consisted of a linear slope starting at 10%B and ending at 100%B over 2.5 min. This method provided a peak capacity of 65, which was sufficient for our analysis of relatively pure compounds. Analysis plates were spun at 1000 rpm for 2 min before final analysis. A 1-µL injection was performed directly from the 96-well analysis plate with no dilution. The detection was accomplished by UV at both 215 nm and 254 nm using an Agilent Micro Flow Cell with 6-mm path length (G1315–60015), and the mass of the target compound was verified by the M + H peak. An external caffeine standard was run in triplicate at the beginning, middle, and end of each time point. The relative standard deviation (RSD) of the replicate external caffeine standards for each plate was <0.5% within each time point, indicating sufficient injection reproducibility.

Data processing and quantitation

The peak height for each compound was measured at 215/254 nm before the study began, and the wavelength, which provided a response in the range of 1–2 AU, was selected to ensure the compound would have both sufficient signal to noise (S/N) in the case of degradation and would also remain within the linear range of the detector. In cases where both 215 nm and 254 nm gave a similar response, 254 nm was selected because of better S/N from reduced baseline noise at that wavelength. Once the wavelength was identified for each compound, the peak area was measured by an optimized ChemStation (Dayton, OH) integration routine and the final result exported to a Microsoft Access database for subsequent processing and correlation.

The initial peak area for each compound was first determined at t₀. All subsequent time points were processed in the same way and used to normalize to the initial t₀ peak area to monitor the relative change in concentration over the course of the study. A freshly prepared 10-mM stock solution of caffeine in DMSO was also analyzed at each time point to account for any drift in the UV detector over the course of the 18-week study. Data were normalized by the following equation:

Normalized peak area = \frac{A_{n} {/ S}_{n}}{A_{o} {/ S}_{o}},

(1)

where A_n and S_n are the peak areas of the targets and caffeine standard, respectively, at the given time point, with t₀ being the initial time point. The RSD of the caffeine standard peak area over the course of the study was <3%, indicating the HPLC system was stable within our requirements. Data are presented as a normalized peak area to t₀ but are a direct measure of the change in concentration over the course of the study. The majority of the results are presented as a best polynomial fit (Igor Pro 6.1; WaveMetrics, Lake Oswego, OR) to the mean peak area for all the study compounds at each time point. Error bars are presented as the standard error of the mean for each data set.

A final analysis of the sample purity at time point 5 was completed to compare the purity of compounds between the exposure and solubilization sets. After time point 5, samples had been stored for 18 weeks and undergone 5 exposure or solubilization cycles. This allowed for a meaningful comparison between the liquid chromatography (LC)–UV purity to verify that the solubilization protocol did not negatively affect compound purity. Purities were measured at UV215 and reported as a percentage of the target peak to the impurity peaks.

Results and Discussion

Validation of study volumes

Consideration of the factors related to the volume of DMSO solution in each tube was important because water uptake by DMSO is a function of both exposure time and the surface-to-volume ratio in the tube. DMSO water uptake in microplates has been shown to be on the order of hours, leading to the potential for significant water uptake from minimal sample handling alone.¹¹ Because this study required different volumes between the single-use temperature and multiuse exposure/solubilization sets, a preliminary validation experiment was conducted to compare the water uptake between the 30-, 150-, and 600-µL volumes in the Matrix storage tubes used for study. These data were necessary to verify that the water uptake at the lower volumes used in the temperature set would not be significantly faster than for the larger volumes used in the multiuse studies. The results, presented in Supplemental Figure S1, were obtained by measuring the weight-% of water in DMSO of uncapped tubes over a 2.5-h time period, which was the anticipated cumulative exposure time for the multiuse exposure/solubilization sets. These data indicated that although the water uptake in 30 µL was roughly fourfold higher than in 150 µL, the total amount of water after 2.5 h was not above historical levels we have seen in our compound store.¹² In addition, because the 30-µL samples were intended only as single use, the total exposure was anticipated to be <1 h, or a water content of around 1%, which was similar to the 150-µL samples after the full 2.5 h. Overall, this confirmed that higher volumes are better to minimize water uptake, but 30 µL used in this study did not result in excessive uptake within our timeframe. We also analyzed the concentrations of a 600-µL control sample from the exposure set after 18 weeks of storage and saw similar results to those presented, giving us further confidence that the sample volume had minimal impact on our study results.

Selection of LC-UV-MS methodology

A variety of techniques for absolute quantitation of library compounds have been reported. Nuclear magnetic resonance (NMR) is routinely identified as the most accurate but also the most expensive and least amenable to automation for larger sample sets. Alternative HPLC detectors such as evaporative light-scattering detection (ELSD), chemiluminescent nitrogen detection (CLND), and charged aerosol detection (CAD) have been shown useful for targeted sets of similar compounds but are less applicable to diverse libraries where absolute errors of 20% to 50% have been reported.^13–16 In addition, these detectors depend on a number of parameters to ensure reproducible introduction of the sample and can be affected by HPLC method conditions. Without careful attention to maintenance, calibration, and sample conditions, these detectors can exhibit drift over time, further limiting the usefulness for a time-based study. As an alternative approach to absolute quantitation or relative purity over time, we decided that monitoring the relative change in concentration of the parent with reference to an external standard would offer sufficient data. This approach would also allow the use of a standard UV detector, which offers a linear response over a wide dynamic range and exhibits minimal drift over time. This provides a more universal approach to analyzing a diverse set of compounds without the use of the individual standards or less reliable detectors.

Temperature study

The goal of the temperature set was to characterize the changes in concentration for the selected library of compounds across an 18-week time period. The samples were all prepared as single-use samples such that any change in the concentration could be related directly to the temperature storage conditions. The results of the temperature set are presented as the mean change in concentration at each time point in Figure 1 . The results indicate that over the course of the study, the mean concentration decreased by 20% for the room temperature conditions compared to a 10% decrease for the frozen compounds. Although this decrease is acceptable for the frozen conditions, it was outside of our criteria for the room temperature conditions, indicating an alternative protocol may be needed once compounds have been stored for more than 18 weeks at room temperature. In the early part of the study, a slight increase in concentration (mean concentration of ~1.03) was noted, but this was within the RSD of the external standard throughout the course of the study and was considered acceptable experimental drift. Further analysis of the raw data indicated that ~85% and 60% of the compounds in the frozen and room temperature storage condition, respectively, showed less than a 20% decrease in concentration over the course of the study.

Fig. 1.

Temperature set. Comparison of best fits to mean normalized peak area for −8 °C (dashed) and 20 °C (solid) storage conditions in the temperature set study with error bars representing standard error (n = 1 for each time point).

It is worth noting that the compounds that showed the greatest change after 18 weeks showed this change at the early time points. For all of the compounds in our study, if the compound experienced a >50% change in concentration, this occurred during the first two time points (specific examples can be seen in Suppl. Fig. S2). Previous estimates indicate that 5% to 10% of library compounds are not soluble in DMSO at library concentrations, whereas an even higher percent may have a significant dependence on temperature.¹⁷ Although chemical instability cannot be completely ruled out using a single LC-MS method, such rapid degradation would also be unlikely across a diverse set and is more consistent with solubility concerns. Tubes that showed the greatest change were visually inspected at the conclusion of the study, with each showing evidence of precipitate, further pointing to solubility being the biggest concern with this sample set.

Exposure study

The exposure set was designed to evaluate the impact of multiple exposure cycles on a single sample tube over the course of the study. As our archive material exists in multiuse storage tubes, understanding the cumulative impact of handling exposure is an important factor. Our results, presented in Figure 2 as the mean concentration at each study time point for all the compounds analyzed, indicate that no measureable effect from the exposure cycles was observed. Although a slight difference was observed for the samples stored at room temperature after five exposure cycles, this effect is minimal when compared to the impact that was observed at different storage temperatures. The data with respect to storage conditions were consistent with the temperature set, indicating that storage temperature affects compound concentration much more than exposure. These data also show that by storing a compound at room temperature to eliminate a true freezing-and-thawing cycle, it does not outweigh the impacts of temperature within our timeframes and exposure cycles. Previous reports have indicated that typically 10 to 20 exposure cycles are needed before a statistically meaningful impact can be seen,⁸ whereas other reports have indicated that <10 cycles have no impact on compound concentration.⁴ Our data seem consistent with these reports for a low number of exposure cycles. It may also reflect the labware/volume-specific issues that may be unique to each study, making comparisons more difficult due to the labware-dependent rate of DMSO water uptake. Careful consideration must be given to approaches that minimize sample freeze-thaw cycles at the cost of increased storage temperatures. One question that still remains unanswered from both our and others’ results is whether the increased wt% water from ambient uptake or the actual temperature cycle associated with thawing has a greater impact on compound concentration and stability. Future studies that characterize the individual effects from sample handling and water uptake would be of value, but the practical nature of sample handling may mean that it is impossible to eliminate water uptake concerns for routine operations.

Fig. 2.

Exposure set. Exposure cycle comparison of average peak area for all study compounds at frozen and room temperatures. Data presented as best fit to mean peak area at each time point and condition with error bars representing standard error (n = 1 for each time point).

Solubilization study

A final study was completed to evaluate the impact of AFA technologies to improve the consistency of the compound concentration over an extended time period. In this set, a single sample was characterized over the course of the study with the addition of an AFA protocol applied before dispensing into the analysis plate. The hope was that this protocol would resolubilize the target compound prior to analysis to mitigate loss of compound due to precipitation. The results of this analysis are presented in Figure 3 as a comparison between the mean peak areas of the solubilization protocols at both frozen and room temperatures across all five time points. From these results, a positive impact on the concentration of the target compound can be noted, especially for the later time points where the concentration increased by an average of 10% for the frozen conditions and 7% for the room temperature conditions. As with the temperature study, the slight increase in mean concentration at the early time points is within the error of the external standard and considered acceptable. This study was consistent with our earlier observations in both temperature and exposure studies where temperature had a far greater impact on final compound concentration than other factors.

Fig. 3.

Solubilization set. Comparison of mean peak areas for solubilization protocols at frozen and room temperature storage conditions across all 18-week storage times. Lines represent best fit to data acquired at specified time points with error bars representing standard error (n = 1 for each time point).

The greater impact of the solubilization protocol on the frozen set is consistent with decreased compound solubility at reduced temperatures, leading to a greater degree of precipitation. The application of the AFA protocol prior to analysis, therefore, helps to reduce precipitated material and increases the effective concentration in solution. Although this seems like a reliable approach, it should be noted we have not characterized the kinetic solubility factors associated with the AFA protocol. Differences between thermodynamic and kinetic solubility have been well documented,¹⁸ and it is possible to super-saturate solutions due to kinetic effects, only to have the compound rapidly (<24 h) precipitate as it reaches the thermodynamic equilibrium. In our results, we analyzed the samples around 2 h after the AFA protocol was applied. This timeframe may be feasible for internal compound management reformatting operations but would be unrealistic from the perceptive of a high-throughput screening (HTS) group where a significant delay between formatting and analysis can occur. These kinetic factors will be important to understand as we evaluate the potential of AFA technologies in our compound management workflow.

Another concern when using acoustic methods for solubilization is the effect on sample purity. Earlier reports have generally shown that methods using both traditional sonication and focused acoustics are relatively soft and show little impact on compound degredation.^9,10 The purity of the compounds was further analyzed over the course of multiple solubilization cycles, as would be required in the compound management environment, to evaluate the combined impact of exposure and solubilization cycles on sample stability. These results (shown as raw data in Suppl. Fig. S3) indicate very little difference between those compounds subjected to the solubilization protocol and those being directly analyzed after storage under frozen conditions, with the mean purities for both conditions being 94%. The data for the room temperature conditions, however, indicate slightly higher purities (mean purity 86% vs. 72%) for the compounds not undergoing the solubilization protocol. This is likely because the protocol is nonselective and will act to resolubilize everything in the tube, including undesired degredants.

From these data, we have seen that AFA is a potential tool to resolubilize library compounds in both frozen and room temperature conditions. Our results are consistent with previous studies that indicate temperature is more important to maximizing compound concentration over storage life, and even with resolubilization protocols, compounds stored at a higher temperatures will have a decreased lifetime. What we remain concerned about and were not able to address in this study with respect to AFA is the kinetic solubility factor. Although it seems that AFA is an approach to resolubilize compounds, the fact that the concentrations may be quite dynamic is likely of greater concern to HTS workflows.

We have been able to conclude three key points from this focused study:

Compound concentration decreased by 20% after 18 weeks of storage at room temperature compared to only 10% for low-temperature conditions.

A limited number of exposure cycles (≤5) had little impact on compound concentration with storage temperature being far more important.

A high-throughput resolubilization protocol using adaptive focused acoustics increased the concentration of compounds under both temperature conditions, but more research is needed to understand kinetic solubility factors.

These data support our hybrid storage approach where newly prepared compounds that have a high demand are stored in a short-term store at room temperature. They also indicate that temperature is a more critical factor than exposure cycles, so caution must be used when trying to minimize freezing and thawing by storage at elevated temperatures. Finally, the data indicate the possibility of increasing the use of focused acoustics for thawing and resolubilization to potentially eliminate the need for short-term room temperature storage.

Footnotes

Acknowledgements

We acknowledge the Amgen Materials Management group for their support of this work. Cheryl Dhlos was instrumental in developing the Covaris solubilization protocol and assisting us with implementation. Conversations with Yaxiong Sun and Amanda Lembke also provided valuable insights into the sample handling operations. Finally, we thank John McCarter and David Semin for thoughtful conversations regarding the project and final manuscript.

Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at .

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Supplementary Material

Please find the following supplemental material available below.

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Characterization of Short-Term Temperature,Exposure,and Solubilization Effects on Library Compound Quality

Abstract

Keywords

Introduction

Materials and Methods

Study design

Sample preparation, storage, and exposure

Sample solubilization protocol

Sample analysis by LC-UV-MS

Data processing and quantitation

Results and Discussion

Validation of study volumes

Selection of LC-UV-MS methodology

Temperature study

Exposure study

Solubilization study

Footnotes

Acknowledgements

References

Supplementary Material