SEE http://www.google.co.in/patents/US20100298257
Example 28
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Chemical Purity Determination by HPLC
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Various HPLC conditions can be used to determine the chemical purity of the compounds disclosed herein. One such example is disclosed above in relation to the thermodynamic aqueous solubility studies. Another example is disclosed below.
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HPLC Conditions:
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- LC: Waters Alliance 2695 Separations Module, Waters 2996 PDA detector and Waters Empower 2 Software (Version 6.00)
- Column: Phenomenex Luna C18(2); 4.6×50 mm; 3 μm
- Flow rate: 1.2 mL/min
- Injection Volume: 10 μL
- Mobile phase: Solvent A: 95% Water with 5% Methanol and 10 mM Ammonium Acetate; pH˜5.3
- Gradient Solvent B: MeOH with 10 mM Ammonium Acetate hold at 0% B 3 min
- 0-47% B 3-4 min
- hold at 47% B 4-10 min
- 47%-74% B 10-11 min
- hold at 74% B 11-13.5 min
- return to 0% B 13.5-13.6 min
- hold at 0% B 13.6-15.5 min
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Under these conditions, the purity of 4, RP-4, and SP-4 was determined to be ˜99.6, ˜99%, and ˜99.5%, respectively. It is noted that higher purities can be realized by optimizing the methods disclosed above.
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Inspection of the XRPD diffractograms shows that the two crystalline single diastereoisomers gave clearly different XRPD patterns. Additionally, there was a clear difference in the melting point of the two crystalline diastereoisomers, with RP-4 having a considerably higher onset than SP-4 (136° C. vs. 94° C.).
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Example 29Additional Separation Methods
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The following SFC separation (conditions listed below) yielded adequate separation of a mixture of the diastereomers, RP-4 and SP-4.
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Preparative Method: Analytical Method: Chiralpak AS-H (2 × 25 cm) SN# 07-8656 Chiralpak AS-H (25 × 0.46 cm) 20% methanol/CO2 (100 bar) 20% methanol/CO2 (100 bar) 50 ml/min, 220 nm. 3 ml/min, 220 nm. Conc.: 260 mg/30 ml methanol, inj vol.: 1.5 ml -
The following SFC separation (conditions listed below) yielded adequate separation of a mixture of the diastereomers, RP-4 and SP-4.
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Preparative Method: Analytical Method: Chiralpak IA(2 × 15 cm) 802091 Chiralpak IA(15 × 0.46 cm) 30% isopropanol(0.1% DEA)/CO2, 40% methanol(DEA)/CO2, 100 bar 100 bar 60 mL/min, 220 nm. 3 mL/min, 220 nm. inj vol.: 2 mL, 20 mg/mL methanol -
TABLE 16 Summary of results from the batch characterization of RP-4, 4, and SP-4. Analysis RP-4 4 SP-4 Proton NMR Single diastereoisomer 1:1 Mixture of Single diastereoisomer diastereoisomers XRPD Crystalline – different Amorphous Crystalline – different DSC from SP-4 Endotherm; 59° C. from RP-4 Endotherm; melt – 136° C. Endotherm; melt – 94° C. TGA No wt loss, No wt loss, decomposition No wt loss, decomposition >240° C. >240° C. decomposition >240° C. IR See above See above See above Aq Solubility 1.58 6.11 5.65 (mg · ml−1) HPLC Purity 96.9% 99.6% 99.5% 40° C./75% RH No form change Deliquescence inside 1.5 h Deliquescence inside 4.5 h 25° C./53% RH — Deliquescence No form change GVS Non-hygroscopic up to 90% — Non-hygroscopic up to 60% RH RH
- Example 27Thermodynamic Aqueous Solubility
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Aqueous solubility was determined by suspending a sufficient amount of compound in water to give a maximum final concentration of ≧10 mg.ml−1 of the parent free-form of the compound. The suspension was equilibrated at 25° C. for 24 hours then the pH was measured. The suspension was then filtered through a glass fiber C filter into a 96 well plate. The filtrate was then diluted by a factor of 101. Quantitation was by HPLC with reference to a standard solution of approximately 0.1 mg.ml−1 in DMSO. Different volumes of the standard, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.
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TABLE 14 HPLC Method Parameters for Solubility Measurements Type of method: Reverse phase with gradient elution Column: Phenomenex Luna, C18 (2) 5 μm 50 × 4.6 mm Column Temperature 25 (° C.): Standard Injections (μl): 1, 2, 3, 5, 7, 10 Test Injections (μl): 1, 2, 3, 10, 20, 50 Detection: 260, 80 Wavelength, Bandwidth (nm): Flow Rate (ml · min−1): 2 Phase A: 0.1% TFA in water Phase B: 0.085% TFA in acetonitrile Time (min) % Phase A % Phase B Timetable: 0.0 95 5 1.0 80 20 2.3 5 95 3.3 5 95 3.5 95 5 4.4 95 5 -
[0306]Analysis was performed under the above-noted conditions on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software vB.02.01-SR1.
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TABLE 15 Aqueous solubility result for RP-4, 4, and SP-4. pH of Unfiltered Sample ID mixture Solubility/mg · ml−1 Comments RP-4 7.12 1.58 Suspension 4 7.03 6.11 Residual solid SP-4 6.88 5.65 Residual solid
Chemical structures of RBV, BOC, TVR, and VRT-127394. Shown are the chemical structures of the anti-HCV drugs RBV {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2,4-triazole-3-carboxamide)} (A), BOC {(1R,2S,5S)-N-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-yl)-3-[(2S)-2(tertbutylcarbamoylamino)-3,3-dimethylbutanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide} (B), TVR {(3S,3aS,6aR)-2-[(2S)-2-[[(2S)-2-cyclohexyl-2-(pyrazine-2-carbonylamino)acetyl]amino]-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1, 2-dioxohexan-3-yl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole-3-carboxamide} (C), and VRT-127394 (R diastereoisomer of TVR) (D).
Blank plasma samples used for matrix effect (ME) assessment and for the preparation of calibration and control samples were obtained from citrated blood (1,850 × g, 10 min, +4°C, Beckman J6B centrifuge) collected from Vaquez disease patients on the occasion of their regular phlebotomy.
The blank plasma used for the preparation of the calibration and quality control (QC) samples was acidified with 10% FA (50 μl of 10% FA added to 950 μl of plasma). The acidification of plasma aims at preventing the conversion of TVR to its epimer VRT-127394 that occurs in vivo and in vitro. (Tibotec-Janssen, personal communication).
Equipment.The LC system used consisted of Rheos Allegro quaternary pumps equipped with an online degasser and an HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) controlled by Janeiro-CNS 1.1 software (Flux Instruments AG, Thermo Fischer Scientific Inc., Waltham, MA). Separations were done on a Hypercarb 3-μm column (2.1 mm ID by 100 mm; Thermo Fischer Scientific) placed in a column oven thermostat regulated at +80°C (HotDog 5090; ProLab GmbH, Reinach, Switzerland). The chromatographic system was coupled to a triple-stage quadrupole quantum mass spectrometer (Thermo Fischer Scientific) equipped with an electrospray ionization (ESI) Ion Max interface and operated with the Xcalibur software package (version 2.0; Thermo Fischer Scientific).
READ
http://www.us.edu.pl/uniwersytet/jednostki/wydzialy/chemia/acta/ac14/zrodla/14_AC14.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291777/
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Filed under: ANALYTICAL Tagged: HPLC, Sofosbuvir
