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High Throughput Determination of Compound Solubility
Dr. Sergio Guazzotti, Product Manager, Chemical Analysis, Nanostream Inc.
Introduction
Figure 1. Nanostream CL System for
high throughput liquid chromatography.
Click to enlarge. |
Compound solubility should be determined during drug lead optimization to aid
in the selection of promising drug candidates prior to biological testing to make
certain that screening results will be meaningful. The evaluations of compound
solubility employing conventional approaches (such as the shake-flask method)
are often inadequate in modern high throughput screening (HTS) environments since
these determinations require large amount of sample in solid form, time, and manpower
and samples are routinely provided at 10 mM in DMSO. It is highly desirable to
establish methodologies that can adapt to these constraints and be integrated
within the activities of the screening laboratory 1-2. Some of the
techniques used for solubility evaluations, such as nephelometry and flow cytometry,
provide adequate throughput, but are not sensitive to compound purity and identity.
Conventional HPLC and LC-MS methods overcome these drawbacks but suffer from their
intrinsic low throughput nature when used in a serial mode. High throughput liquid
chromatography offers the advantages of a separation-based approach (such as HPLC
and LC-MS) but provides 10 times more throughput than conventional HPLC while
reducing sample consumption,
solvent usage and waste generation.
Materials and Methods
Equipment: Nanostream CL System equipped with a Brio Cartridge 4207 with 24 parallel
columns (80ࡦ.5 mm (i.d., equiv.)) (Figure 1)
Standards: Sets of calibration standards were prepared for 24 compounds at different concentrations (in a 50:50 ACN:H20 solvent). A maximum standard concentration of 500 μM was selected to keep the amount of DMSO co-solvent in all samples and standards below a 5% (v/v) when working with stock solutions provided at 10 mM in DMSO. Standards were then added to the appropriate wells of a 384-well plate. The plate was covered with a heat seal foil and transferred to the Nanostream CL System for analysis.
Figure 2. Chromatograms obtained simultaneously
for 24 different compounds employing the Nanostream CL System. Analytical
Conditions: Stationary phase: C18; Mobile phase A: 95:5 H20/ACN w/0.1% formic
acid; Mobile phase B: ACN w/0.08% formic acid; Flow rate: 12.5 mL/min (for
each column); Gradient: t = 0 to 1.0 min, 5% B; t = 1.0 to 6.0 min, 5 to
95% B; t = 6.0 to 7.0 min, 95% B. UV absorbance detection: L = 254 nm. Click
to enlarge. |
Sample Preparation: 95 μL of a buffer of desired pH was added to the appropriate wells of a 384-well plate. An additional 5 μL of each compound at a concentration of 10 mM (in DMSO) was added to the corresponding wells. The plate was shaken for 90 minutes and centrifuged at 4000 rpm for three minutes. Accurate and reproducible control of the depth of the autosampler needles permitted the sampling of the supernatant solution without perturbing the precipitate, thus avoiding the need for sample filtration.
Data Analysis: Compound solubility values were evaluated from interpolation of the corresponding compound peak area obtained from a solution prepared with appropriate buffer within the corresponding external standard calibration curves (peak area vs. concentration).
Results and Discussion
Sample chromatograms obtained simultaneously for the 24 samples investigated are shown in Figure 2. Data analysis was performed using Nanostream's advanced analysis software to automate the analysis of samples and generation of calibration curves. Linear regressions were evaluated for all standard curves yielding R2 values between 0.98 and 1.0. Aqueous solubility values for the 24 samples compared favorably with results obtained by other traditional methods. Figure 3 shows the result of the evaluation of aqueous solubility at four different pHs for phenazopyridine and piroxicam samples.
Conclusions
Figure 3. Solubility values at different
pH values obtained for a phenazopyridine sample (top panel) and a piroxicam
sample (bottom panel) employing the Nanostream CL System. Values are compared
with reported values3,4. Literature values adapted from cited manuscripts.3,4.
Click
to enlarge. |
These results demonstrate the utility of the Nanostream CL System for the high
throughput determination of compound solubility. Significantly lower volumes of
mobile phase were employed for these determinations with the Nanostream CL System
than with traditional HPLC methods. For example, for the evaluation of 24 compounds,
employing a 4-point external standard calibration curve and duplicate analysis,
a total of 33 mL of mobile phase was consumed with the Nanostream CL System whereas
a traditional HPLC system required 2.6 L for the same determinations. The high
throughput capabilities provided by the Nanostream CL System allowed for the generation
of calibration curves for 24 compounds (4-point calibration curve (duplicate analysis))
and duplicate solubility measurements for each sample (240 separations) in approximately
2 hours (not including incubation time). Employing similar conditions, a total
of approximately 1900 samples could be evaluated per week (4-point calibration
curve, singlet (1152 separations per day)). Minimal sample quantities (5 μL
of 10-mM solutions in DMSO for this study) were required for all these determinations.
References
1. Kassel, D. B. (2005). High throughput strategies for in vitro ADME assays: How far can we go? In W. A. Korfmacher (Ed.), Using mass spectrometry for drug metabolism studies. Boca Raton, FL: CRC Press.
2. Bevan, C.D., and Lloyd, R.S. (2000). A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtiter plates. Analytical Chemistry, 72, 1781-1787.
3. Avdeef, A. (2003). Absorption and drug development: Solubility, permeability, and charge state. Hoboken, NJ: John Wiley & Sons.
4. Wexler, D. S., Gao, L., Anderson, F., Ow, A., Nadasdi, L., McAlorum, A., et al. (2005). Linking solubility and permeability assays for maximum throughput and reproducibility. Journal of Biomolecular Screening, 10(4), 383-390.
Dr. Sergio Guazzotti may be contacted at sergio.guazzotti@nanostream.com
or by phone at 626-351-8200 x6716.
AT A GLANCE
• Evaluations of compound solubility employing conventional approaches are often inadequate in high-throughput screening (HTS) environments
• High-throughput liquid chromatography offers the advantages of a separation-based approach, but provides 10 times more throughput than conventional HPLC
• Aqueous solubility values for the 24 samples compared favorably with results obtained by other traditional methods
• Significantly lower volumes of mobile phase were employed for determinations with the Nanostream CL System than with traditional HPLC methods
ONLINE
For additional information on the technologies discussed in this article, see
Laboratory Equipment magazine online at www.LaboratoryEquipment.com
or the following Web site:
• www.nanostream.com
Laboratory Equipment
Advantage Business Media
Rockaway, NJ, 07866
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