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An application of Fourier transform infrared (FT-IR) spectroscopy equipped with an attenuated total reflectance (ATR) probe for in-line monitoring of a hydrochloride (HCl) salt formation process of 4-{1-methyl-2-piperidin-4-yl-4-[3-(trifluorometryl)phenyl]-1H-imidazol-5-yl}-N-[(1S)-1-phenylethyl]pyridine-2-amine (freebase), an active pharmaceutical ingredient as a P38 MAP kinase inhibitor, is described. The freebase forms both mono- and bis-HCl salts due to its structural features. The mono-HCl salt is the desired product but the bis-salt is an impurity. The key to maximizing the product yield and minimizing the impurity level is to monitor the salt-forming reaction and to terminate it at the correct HCl charge amount. The process analytical technology (PAT) provided real-time data for process control and overcame the limitations that had been previously encountered by other analytical instrumentations, such as high-performance liquid chromatography and titration. Two qualitative approaches for reaction endpoint determination were employed. In the first approach, changes in the concentration of the freebase and bis-salt were monitored via the first derivative concentration profiles. The flat point in the freebase profile and the rise in the bis-salt profile were used as a detection bracket for the endpoint of HCl charging. In the second approach, principal component analysis (PCA) was used to classify the status of the process based on a spectral library consisting of spectra collected around the endpoint. Results indicated that both methods provided adequate accuracy for endpoint control in a small window between 1.0 and 1.05 HCl to freebase mole ratio. Both methods were used to support a scaled up process. Three batches of MAP mono-HCl salt formation were successfully controlled and prepared.

A novel approach for the potential on-line determination of the enantiomeric excess (ee) of an M3 antagonist drug substance combining attenuated total reflectance infrared (ATR-IR) spectroscopy, guest-host complexes, and chemometric data analysis is described. Chiral recognition through a formation of diastereomeric complexes was measured by ATR-IR. Small changes on the IR spectra reflect the interaction between the guest (M3) and host (chiral selector). These changes are measured as a function of M3 enantiomer excess. The standard error of prediction is 1.3 ee%. The prediction results based on the IR method were in good agreement with the gravimetric method. The robustness of the calibration model was evaluated by varying the concentration of the chiral selector, the pH of the solution, and the organic solvents. The stability of the calibration model was also demonstrated through measuring different sets of samples on different days. Chirality, 2006. © 2006 Wiley-Liss, Inc.

A large number of samples can be generated during pharmaceutical process development. Fast separation for these samples is usually challenging due to the complexity of sample matrix, which requires high efficiency as well as high speed. Monolithic columns (E. Merck, Germany) were investigated as a possible tool for reducing separation time in reversed-phase HPLC without significantly sacrificing efficiency or resolution. Both van Deemter plots and separations of alkyl benzenes and in-process samples showed that monolithic columns were suitable for fast separations without significantly compromising resolution. Practical parameters including the pressure drop, retention factor, selectivity, and tailing factor of monolithic columns (Chromolith type) were compared to those of conventional YMC 150 mm × 4.6 mm (3-μm particles) and 250 mm × 4.6 mm (5-μm particles) packed columns. The batch-to-batch reproducibility of the 100 mm × 4.6 mm Chromolith columns from five randomly ordered batches was also compared to the 250 mm × 4.6 mm YMC particle-packed columns. Fast and efficient separations of complicated process samples including crude drug substances, reaction mixtures, and crystallized mother liquors were demonstrated for both monolithic columns and conventional packed columns. The analysis times were decreased by three to seven times on the coupled monolithic columns, while maintaining the comparable resolution to typical 5-μm particle-packed 250 mm × 4.6 mm columns.

This paper reports a comparison study of the difference between Chiralpak AD-H and AD columns in enantioseparation of dihydropyrimidinone (DHP) acid and its methyl ester under normal phase LC conditions. Unlike those of the AD phase, the van't Hoff plots of retention factors for DHP acid on the AD-H phase were linear. The cyclic van't Hoff plots of selectivity factors for DHP acid on the AD-H phase were non-linear and slightly non-superimposable. No conformational transition was observed on the AD-H phase in the whole temperature range. A single-step temperature program on the AD-H phase showed that the selectivity factors of DHP acid only increased approximately 1.7% in 24 h (versus approximately 50% on the AD phase). For DHP ester, the single-step temperature program showed that the selectivity factors on the AD-H phase remained the same in 24 h while those on the AD phase increased around 3.1%. The enantioselectivity of DHP acid on the AD-H phase was lower than that on the AD phase while the enantioselectivity of DHP ester on the AD-H phase was higher than that on the AD phase. The resolution of DHP acid on the AD-H phase was about the same as that on the AD phase while the resolution of DHP ester on the AD-H phase was much higher than that on the AD phase. The results of DHP acid are opposite of what the vendor suggested while the results of DHP ester are the same as the vendor's application notes. This indicates that the differences between Chiralpak AD-H and AD columns are not only in their particle size, but also in the solvated conformations.

The possible mechanisms for the chiral recognition of 2(S)-(3,5-bis-trifluoromethyl-phenyl)-2-[3(S)-(4-fluorophenyl)-4-(1H-[1,2,4]triazol-3-ylmethyl)-morpholin-2(R)-yloxy]-ethanol (compound A) and its enantiomer with native gamma-cyclodextrin (gamma-CD) were investigated using capillary electrophoresis (CE), reversed-phase liquid chromatography (RPLC), proton (1H), fluorine (19F) and carbon (13C) nuclear magnetic resonance spectroscopy (NMR), electrospray mass spectrometry (ESI-MS) and circular dichroism (CD). All experiments provided clear evidence of the formation of diastereomeric complexes between the enantiomers and gamma-CD. Proton, fluorine and carbon NMR spectra suggested that both aromatic rings, with mono-fluoro and bis-tri-fluoro functional groups, on the guest molecule were partially included into the cavity of the gamma-CD. ESI-MS spectra indicated that the diastereomeric complexes have a 1:1 stoichiometric ratio. The binding constants of the diastereomeric complexes obtained by CE, RPLC and CD were compared. The effects of the gamma-CD concentration, organic modifiers and temperature on the CE-chiral separation were also investigated.

The simultaneous enantioseparation of a basic drug compound, 2(R)-N-[1-(6-amino-pyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenyl-acetamide (M3), and its chiral acidic intermediate, (R,R) 1-(2,2-difluorocyclopentyl)-phenylacetic acid (MA), was investigated by capillary electrophoresis (CE) with various cyclodextrins (CDs). After an initial screening, a single-isomer CD, octakis(2,3-diacetyl-6-sulfo)-gamma-CD (ODAS-γ-CD), was selected for further method development based on separation selectivity and the peak migration time. Other method parameters such as pH of background electrolyte (BGE), type of capillary, temperature, and organic modifier were varied in order to optimize the method. The optimal method was validated in terms of linearity, sensitivity, precision, ruggedness, and specificity. A mixture of enantiomers of M3 and MA were spiked into a matrix of reagents used for the synthetic step and the resulting solution was evaluated by the optimized CE-chiral method. The results indicate both pairs of M3 and MA enantiomers were free of interference from the reaction matrix. Therefore, the feasibility of utilizing the method to monitor possible enantio-conversion during the synthetic process was demonstrated.

The enantioseparation of a weakly basic pharmaceutical drug substance is investigated using sulfated-β-CD as a chiral selector. The enantiomers are separated at acidic pHs in an anodic flow due to complexation with sulfated-β-CD, while under an electroosmotic flow (EOF) counter-current. At basic pHs, the EOF prevails and the analytes migrate toward the cathode, where improved separations can be obtained along with an apparent reversal of migration order. The optimal enantioseparation of the test compound is systematically explored by varying important factors, such as pH, concentration of sulfated-β-CD, temperature, and addition of organic modifier. The implications of the results on general enantioseparation of weakly basic pharmaceutical compounds are discussed.

The chiral separation of an M3 antagonist was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins and by reversed-phase liquid chromatography with derivatized cellulose, derivatized amylose, and two protein stationary phases. Operational parameters for each technique, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH and type, organic modifiers, injection mode and temperature were varied in order to achieve a desired elution order and to meet a 0.1% limit of quantitation (LOQ) criteria. Based on the advantages and disadvantages of each technique, a practical CE method using sulfated gamma-cyclodextrin was selected. The method was validated in terms of linearity, LOQ, accuracy, ruggedness and precision.

The feasibility for separation of positional isomers using capillary electrochromatography (CEC) was investigated. Two groups of neutral isomers, trifluoroacetophenone and bromobenzonitrile, were separated by CEC. Effects of selected parameters, such as mobile phase pH, ionic strength, temperature, organic modifier, buffer type, diluent type, voltage, and column length, were studied using bromobenzonitrile isomers to elucidate the separation mechanisms. In parallel, the separation of the isomers was also performed by high-performance liquid chromatography (HPLC) to serve as a comparison. It was found that the retention time behavior as a function of acetonitrile concentration in CEC was similar to that observed in HPLC. The effect of mobile phase pH has a greater impact with CEC than with HPLC due to the change in electroosmotic flow. The effect of temperature with liquid chromatography showed a linear Van't Hoff plot, while deviations from linearity were noted with CEC. The optimized CEC method was valid...

The stereospecific interaction of a neutral probe molecule, acetonide, with immobilized α1-acid glycoprotein (AGP) was investigated. Enantioselectivity was found to be influenced by the choice of organic modifier, temperature, and pH. These parameters could be varied to the extent that a reversal of elution order could be induced. Our studies found that the role of hydrogen bonding in the chiral discrimination of acetonide was minimal. An inclusion mechanism is proposed with the investigated parameters affecting the access to the binding sites either through induced conformational changes or steric hindrance.

The chiral recognition mechanism for a derivatized polysaccharide chiral stationary phase (CSP) under reversed phase conditions was investigated (Chiralcel OJ-R). While enantiorecognition mechanisms have been proposed for these types of phases under normal phase conditions, it is unlikely that they would apply to reversed phase conditions due to the high polarity of the eluent. Indandiol was utilized as the main probe. The effect of organic modifier, eluent anion, and temperature on enantioselectivity for indandiol was determined. Additionally, a number of structurally related probes were also investigated to define the role of various functional groups on enantioselectivity. The investigation revealed that hydrogen bonding did not have a gross direct effect on enantiorecognition but may contribute through it's effect on the tertiary structure of the CSP. It appears that inclusion is the major contributor to enantioselectivity with the size and shape of the probe being the determinant as to the extent of ...

Separations of five synthetic heptadecapeptides from the p-loop region of p21 Ras proteins, differing mostly in a single amino acid at the same position, were investigated. The studies were performed at low pHs in polyacryamide-coated capillaries, where the peptides were slightly positively charged. CZE, without surfactants, failed to separate all the peptides. Separations by MEKC employing an anionic surfactant sodium dodecyl sulfate (SDS), a neutral surfactant Tween 20 and a cationic surfactant cetyltrimethylammonium bromide (CTAB) provided results with varying degrees of success. Tween 20, slightly improved the separation while CTAB resulted in almost baseline separation of all the peptides. The effects of applying acetonitrile to MEKC were also examined. The separation results are analyzed with regard to the properties of the peptides and the surfactants. Implications of the results on peptide-micelle interactions and general aspects of peptide separation by MEKC are discussed.

Due to the high method variability (typicallyor = 0.5%, based on a literature survey and internal Merck experience) encountered in the HPLC weight percent (%) assays of various active pharmaceutical ingredients (APIs), it is proposed that the routine use of the test in stability studies should be discouraged on the basis that it is frequently not sufficiently precise to yield results that are stability-indicating. The high method variability of HPLC weight % methods is not consistent with the current ICH practice of reporting impurities/degradation products down to the 0.05% level, and it can lead to erroneous out-of-specification (OOS) results that are due to experimental error and are not attributable to API degradation. For the vast majority of cases, the HPLC impurity profile provides much better (earlier and more sensitive) detection of low-level degradation products. Based on these observations, a Quality-by-Design (QbD) approach is proposed to phase out the HPLC weight % assay from routine API stability testing protocols.

An on-line fiber-optic near-infrared spectroscopic method has been developed for real-time monitoring of distillate composition as a reliable way of assessing the degree of a solvent switch process from methanol and n-propanol to isopropyl acetate. Multivariate statistical methods of Multiple Linear Regression and Partial Least Squares were evaluated for quantitative determination of the total alcohol concentrations in the distillate samples. Robust models were obtained by incorporating temperature variance into the calibration set. Reliable prediction of total alcohol concentration was successfully achieved by near-infrared spectroscopy as judged by a comparison with gas chromatography data.

A capillary electrophoretic ion analysis method was developed for monitoring esterification of sulfuric acid by ethanol to form monoethylsulfate. Since the analytes of interest have no chromophores, detection was performed utilizing indirect photometric detection. Several background electrolyte systems were evaluated to optimize the efficiency of the separation. The method was more sensitive and specific in comparison to alternative techniques. Pseudo-first-order rate constants for the esterification reaction were determined as a function of temperature and the activation energy was calculated.

A capillary electrophoretic method for the determination of a residual alkyl quaternary amine, tetra-n-butylammonium ion (TBA+), in bulk drug and process streams was developed. Since the analyte does not have a chromophore, detection was performed utilizing indirect photometric detection. The influence of the quinine, tetrahydrofuran and sodium acetate concentrations and pH app in the background electrolyte solution upon the efficiency of the separation and effective electrophoretic mobility of both TBA+ and electro-osmotic flow were studied. The ranges in which the various parameters were examined had considerable effects upon both the efficiency and the effective electrophoretic mobility of the electro-osmotic flow; however, the effective electrophoretic mobility of TBA+ was not significantly affected. The optimized method was validated in terms of detector linearity, sensitivity, precision and accuracy.

Indinavir is a protease inhibitor which possesses five chiral centers. Enantioseparation of indinavir and its enantiomer was performed on an amylose type stationary phase, Chiralpak® AD, under normal-phase HPLC and subcritical fluid chromatography conditions. Under the utilized chromatographic conditions, it is believed that the leading interactions are hydrogen bonding between one or both hydroxyl groups of the solute with the carbonyl group of the carbamate. An inclusion mechanism appears to control the chiral recognition. The effect of various modifiers, pressure, and temperature were investigated.

This work describes the high-performance liquid chromatographic (HPLC) methodology used for the analysis of an oxytocin antagonist (L-368,899). The chromatography separates the drug from its major impurity (exo-isomer), four synthetic intermediates and two key raw materials. The method development process, which was required to achieve baseline separation of the eight components, reveals the subtleties and complexities involved in chromatographic method development. The optimized LC method was validated and found to be rugged and reproducible for the impurity profiling of this oxytocin antagonist drug.

MK-0507 (Dorzolamide HCl, (4S,6S)-4-Ethylamino-5,6-dihydro-6-methyl-4H-thieno-[2,3-b] thiopyran-2-sulfonamide 7,7-dioxide hydrochloride) is the first topically active, water-soluble, carbonic anhydrase inhibitor to be developed for the treatment of glaucoma and ocular hypertension. Dorzolamide HCl is an effective and well tolerated agent as monotherapy for patients who cannot tolerate ophthalmic beta-blockers, and for those who need add-on therapy to beta-blockers.1 The steps taken in the development and validation of a fast and rugged reverse-phase HPLC method for the analysis and quality assessment of MK-0507 drug substance and acetamidosulfonamide intermediate are described. Four columns were used during the development: Du Pont Zorbax C-18, Rainin Microsorb C-8, Perkin Elmer CR-C8 and YMC4. The last two columns showed excellent resolution, peak shape, and precision. The selected HPLC method for acetamidosulfonamide, using the Perkin Elmer CR-C8 column, was validated with respect to linearity,...

Investigations into the mechanistic aspects of the stereospecific interaction of the four optical isomers of aminoindanol on a silica based crown ether column were performed. The nature and concentration of the mobile phase's counteranion affected the hydrophobic interaction but had little effect on the inclusion interaction. Minimal changes in the separation factor of the enantiomers were observed in the pH range of 1-5.2, but a minimum in the capacity factor was observed at pH 3.75. Van't Hoff plots indicated a high entropy and a positive enthalpy at pH 5.2, while a lower entropy and a negative enthalpy were observed at and below pH 3.75. Hill plots indicated that there were more active binding sites at pH 3.0 as compared to pH 1.0 and that the binding ratio of aminoindanol to active sites was also greater. Apparently at higher pH values, as the silica becomes deprotonated, there is an additional electrostatic interaction between the protonated aminoindanol and the deprotonated silica sites

The synthesis of pharmaceutical products frequently involves the use of reactive reagents and the formation of intermediates and by-products. Low levels of some of these may be present in the final drug substance and drug product as impurities. Such chemically reactive impurities may have at the same time the potential for unwanted toxicities including genotoxicity and carcinogenicity and hence can have an impact on product risk assessment. This paper outlines a procedure for testing, classification, qualification, toxicological risk assessment, and control of impurities possessing genotoxic potential in pharmaceutical products. Referencing accepted principles of cancer risk assessment, this document proposes a staged threshold of toxicological concern (TTC) approach for the intake of genotoxic impurities over various periods of exposure. This staged TTC is based on knowledge about tumorigenic potency of a wide range of genotoxic carcinogens and can be used for genotoxic compounds, for which cancer data are limited or not available. The delineated acceptable daily intake values of between approximately 1.5 microg/day for approximately lifetime intake and approximately 120 microg/day for < or = 1 month are virtually safe doses. Based on sound scientific reasoning, these virtually safe intake values do not pose an unacceptable risk to either human volunteers or patients at any stage of clinical development and marketing of a pharmaceutical product. The intake levels are estimated to give an excess cancer risk of 1 in 100,000 to 1 in a million over a lifetime, and are extremely conservative given the current lifetime cancer risk in the population of over 1 in 4 (http://seer.cancer.gov/statfacts/html.all.html). The proposals in this document apply to all clinical routes of administration and to compounds at all stages of clinical development. It is important to note that certain types of products, such as those for life-threatening indications for which there are no safer alternatives, allow for special considerations using adaptations of the principles outlined in this paper.

The possible mechanisms for the chiral recognition of 2-(R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide (RR-M3), and its enantiomer (SS-M3) with octakis(2,3-di-O-acetyl-6-sulfo)-gamma-cyclodextrin (ODAS-gamma-CD) and octakis(6-sulfo)-gamma-cycopdextrom enantiomer; (OS-gamma-CD), were investigated using capillary electrophoresis (CE), proton ((1)H), fluorine ((19)F) and carbon ((13)C) nuclear magnetic resonance spectroscopy (NMR), and infrared (IR) spectroscopy. Clear evidence for the formation of diastereomeric complexes between the enantiomers and the two CDs was observed. NMR spectra suggest that the phenyl and difluorocyclopentyl rings are involved in the complexation. The phenyl ring on the guest molecule is deeply penetrated into the cavity of OS-gamma-CD, but it is not included into the cavity of ODAS-gamma-CD. The continuous variation plots built based on the (1)H NMR and IR spectra indicate a 1:1 complex stoichiometric ratio of the M3 enantiomers for both CDs. The affinity of the enantiomers for the two CDs is opposite.

Rofecoxib is a highly active and selective cyclo-oxygenase II inhibitor. A stability-indicating method for the assay of rofecoxib has been developed using reverse-phase high-performance liquid chromatography (HPLC). Stress testing of rofecoxib was conducted during the method development and validation. HPLC analysis of rofecoxib solutions stressed under alkaline and photolytic conditions revealed the presence of several degradates. Two main degradates were determined to be the cyclization product formed by photo-cyclization and the dicarboxylate formed by ring opening in the presence of base and oxygen. The identities of these degradates were confirmed by comparison of UV spectra and HPLC retention time with the independently synthesized products. The mechanistic pathways for the formation of these degradates are discussed. Further improvement of the HPLC method's ruggedness has been made based on these studies.

Enantioseparations of basic pharmaceutical compounds were investigated using different types of sulfated cyclodextrins as chiral selectors. A general strategy for method development was described, together with enantiomeric separations of a number of pharmaceutical related compounds. Based on this strategy, systematic method development approaches for several selected compounds were performed by modifying method parameters, such as the concentration of the chiral selectors, buffer pH, type of organic modifiers, buffer type, temperature and applied voltage. The results of the investigation elucidated the separation mechanism. Many practical aspects were also discussed through several specific examples in order to demonstrate how to develop and validate a precise, sensitive, accurate and rugged separation.

Two low level impurities in 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-methyl-2(1H)-pyridinone drug substance (L-696,229) have been isolated by a combination of preparative HPLC, solid-phase extraction and liquid—liquid extraction. They were identified as 3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-6-(2-phenylethyl)-2(1H)-pyridinone (I) and 6,6′-(2-phenyl-1,3-propanediyl)bis[3-[2-(2-benzoxazolyl)ethyl]-5-ethyl-2(1H)-pyridinone] (II) by mass spectrometry and by their 13 C and 1 H-NMR spectra.

We have developed an FT-IR method, using a Spectra-Tech Monit-IR 400 systems, to monitor off-line the completion of a reaction in real-time. The reaction is moisture-sensitive and analysis by more conventional methods (normal-phase HPLC) is difficult to reproduce. The FT-IR method is based on the shift of a diazo band when a conjugated beta-diketone is transformed into a silyl enol ether during the reaction. The reaction mixture is examined directly by IR and does not require sample workup. Data acquisition time is less than one minute. The method has been validated for specificity, precision and accuracy. The results obtained by the FT-IR method for known mixtures and in-process samples compare favorably with those from a normal-phase HPLC method.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

N-Methoxyphenyl-2-azetidinones are dearylated under mild conditions via anodic oxidation.

Osteryoung square-wave voltammetric analysis is a rapid technique that can be used to determine nicarbazin residues in chicken tissue. The method is reproducible and can be applied to samples containing as little as 1–10 μg/g nicarbazin. This level of sensitivity is more than adequate for meeting governmental regulations. Osteryoung square-wave voltammetry is significantly faster than classical differential pulse polarography.

N-Methoxyphenyl-2-azetidinones are dearylated under mild conditions via anodic oxidation.