Effect of pressure on secondary structure of proteins under ultra high pressure liquid chromatographic conditions.

There are several spectroscopic techniques such as IR and CD, that allow for analyzing protein secondary structure in solution. However, a majority of these techniques require using purified protein, concentrated enough in the solution, to produce a relevant spectrum. Fundamental principles for the usage of reversed-phase ultra high pressure liquid chromatography (UHPLC) as an alternative technique to study protein secondary structures in solution were investigated. Several "model" proteins, as well as several small ionizable and neutral molecules, were used for these studies. The studies were conducted with UHPLC in isocratic mode, using premixed mobile phases at constant flow rate and temperature. The pressure was modified by a backpressure regulator from about 6000psi to about 12,000psi. It was found that when using a mobile phase composition at which proteins were fully denatured (loss of alpha-helix secondary structure), the retention factors of the proteins increased upon pressure increase in the same manner as non-proteins. When using a mobile phase composition in which proteins were not fully denatured, it was observed that the retention factors of the proteins displayed a much steeper (by one order of magnitude) increase in retention upon pressure increase. It was concluded that in a mobile phase in which the protein is not initially fully denatured, the increase of pressure may facilitate the folding back of the protein to its native state (alpha-helix secondary structure). The impact of different mobile phase compositions on the denaturation of the proteins was studied using CD (Circular Dichroism). Moreover, the effect of flow rate on retention of proteins and small molecules was studied at constant pressure on the different pore size silicas and the impact of internal frictional heating was evaluated.

Investigation of Metformin HCl lot-to-lot variation on flowability differences exhibited during drug product processing.

The purpose of this study was to determine the cause for flowability difference observed during drug product processing when different Metformin HCl drug substance batches of varying age were used. It was found that the lead time (age) between the final step (milling) in the manufacturing process of the Metformin HCl drug substance could be a factor. The lead time had an impact on flowability of Metformin/excipient blends during drug product processing even though these batches had no apparent differences in their release specifications. To study and understand the aging effect, two batches of Metformin HCl manufactured at different periods of time were selected. The surface energy values obtained by the density functional theory (DFT) method together with X-ray diffraction patterns, thermally stimulated current measurements, and dynamic vapor sorption isotherms indicated that the freshly manufactured Metformin HCl material contains detectable amounts of surface crystal defects, but are absent in aged sample, which could be the cause of flowability differences of Metformin/excipient blends observed during the drug product processing. Having identified the cause for different flow behavior, a method to destroy these defects was designed and the issue was resolved by rapid aging of Metformin HCl under humidity at room temperature.

Combined application of high resolution and tandem mass spectrometers to characterize methionine oxidation in a parathyroid hormone formulation.

Identification and monitoring of degradation products is a critical aspect of drug product stability programs. This process can present unique challenges when working with complex biopharmaceutical formulations that do not readily lend themselves to straightforward HPLC analysis. The therapeutic 34 amino acid parathyroid hormone fragment (PTH1-34) contains methionine (Met) residues at positions 8 and 18. Oxidation of these Met residues results in reduced biological activity and thus efficacy of the potential drug product. Here, we present an effective approach for the identification of PTH1-34 oxidation products in a drug product formulation in which the stability indicating method used non-MS compatible HPLC conditions to separate excipients, drug substance and degradation products. High resolution and tandem mass spectrometers were used in conjunction with cyanogen bromide (CNBr) mediated digestion to accurately identify the oxidation products observed in an alternative MS compatible HPLC method used for drug substance analysis. All anticipated CNBr digested peptide fragments, including both oxidized and nonoxidized peptide fragments, were positively identified using TOF MS without the need for additional enzymatic digestion. Once identified, the oxidation products generated were injected onto the original non-MS compatible HPLC drug product stability indicating method and the respective retention times were confirmed. This allowed the oxidative stability of different formulations to be effectively monitored during the solid state stability program and during variant selection.

Evaluation of transmission and reflection modalities for measuring content uniformity of pharmaceutical tablets with near-infrared spectroscopy.

This paper examines how one may assess spectral changes with instrument configuration (or composition), in combination with the spectral changes in the measurement that are caused by experimental effects, and subsequently select an appropriate measurement modality for tablet content uniformity determination with near-infrared (NIR) spectroscopy. Two NIR spectrometers furnished with three configurations in the sample measurement interface were evaluated. One spectrometer, Bruker MPA (multiple purpose analyzer), was equipped with two measurement modalities, diffuse transmission (DT) and diffuse reflection based on integrating sphere optics (DR/IS). The other spectrometer, Bruker StepOne, was equipped only with diffuse reflection mode based on a fiber-optic probe (DR/FO). The data were collected with each of the configurations for the tablets associated with two dosage strengths differing significantly in diameter and thickness. Spectral diagnosis was performed in terms of sensitivity and selectivity. The signal-to-noise ratio computed for the data collected with the DT and DR/IS spectrometers was approximately an order of magnitude greater than that computed for the DR/FO spectrometer. The net-analyte-signal-based selectivity analysis of NIR spectra associated with the sample tablet and the placebo tablet indicated that both transmission and reflection mode provided similar selectivity when the optimal spectral range was chosen. A partial least squares (PLS) calibration model was developed for each data set. The overall standard error of calibration for each DT and DR/IS measurement was approximately 0.3% in weight for each strength, significantly better than the value of 1.0% in weight produced by the DR/FO measurement. This result was consistent with the sensitivity analysis based on spectral noise characterization. The poor analytical performance of the DR/FO spectrometer was attributed to the small illumination spot size of the reflection probe and thus the sensitivity of the measurements to the tablet engraving. The PLS analysis and spectral diagnostics both showed that transmission and reflection modes based on the Bruker MPA provided similar measurement accuracy for each strength. However, the robustness study clearly revealed that the transmission mode would be more robust than the reflection mode when there is considerable variability in the chemical composition and physical properties of tablets.

The use of ultra high-performance liquid chromatography for studying hydrolysis kinetics of CL-20 and related energetic compounds.

Ultra high-performance liquid chromatography (UHPLC) utilizes columns packed with sub-2-mum stationary-phase particles and allows operation with pressures of up to 15,000 psi to yield increased resolution, speed, and sensitivity versus conventional HPLC. This promising new technology was used for the analysis of energetic compounds (RDX, HMX and CL-20) and a selective method was developed on an Acquity UPLC. A fast UHPLC method was applied to determine alkaline hydrolysis reaction kinetics of major energetic compounds. Activation energies of alkaline hydrolysis reaction for CL-20, RDX and HMX were comparable to those in literature, however they were determined in a shorter amount of time due to the speed of analysis of the chromatographic method. The use of liophilic salts (KPF(6)) as mobile-phase additives for the enhancement of separation selectivity of energetic compounds was demonstrated.

Content uniformity determination of pharmaceutical tablets using five near-infrared reflectance spectrometers: a process analytical technology (PAT) approach using robust multivariate calibration transfer algorithms.

Near-infrared calibration models were developed for the determination of content uniformity of pharmaceutical tablets containing 29.4% drug load for two dosage strengths (X and Y). Both dosage strengths have a circular geometry and the only difference is the size and weight. Strength X samples weigh approximately 425 mg with a diameter of 12 mm while strength Y samples, weigh approximately 1700 mg with a diameter of 20mm. Data used in this study were acquired from five NIR instruments manufactured by two different vendors. One of these spectrometers is a dispersive-based NIR system while the other four were Fourier transform (FT) based. The transferability of the optimized partial least-squares (PLS) calibration models developed on the primary instrument (A) located in a research facility was evaluated using spectral data acquired from secondary instruments B, C, D and E. Instruments B and E were located in the same research facility as spectrometer A while instruments C and D were located in a production facility 35 miles away. The same set of tablet samples were used to acquire spectral data from all instruments. This scenario mimics the conventional pharmaceutical technology transfer from research and development to production. Direct cross-instrument prediction without standardization was performed between the primary and each secondary instrument to evaluate the robustness of the primary instrument calibration model. For the strength Y samples, this approach was successful for data acquired on instruments B, C, and D producing root mean square error of prediction (RMSEP) of 1.05, 1.05, and 1.22%, respectively. However for instrument E data, this approach was not successful producing an RMSEP value of 3.40%. A similar deterioration was observed for the strength X samples, with RMSEP values of 2.78, 5.54, 3.40, and 5.78% corresponding to spectral data acquired on instruments B, C, D, and E, respectively. To minimize the effect of instrument variability, calibration transfer techniques such as piecewise direct standardization (PDS) and wavelet hybrid direct standardization (WHDS) were used. The PDS approach, the RMSEP values for strength X samples were lowered to 1.22, 1.12, 1.19, and 1.08% for instruments B, C, D, and E, respectively. Similar improvements were obtained using the WHDS approach with RMSEP values of 1.36, 1.42, 1.36, and 0.98% corresponding to instruments B, C, D, and E, respectively.

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography.

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation-desolvation equilibria in the mobile phase, also known as "chaotropic effect". With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF6, NaClO4, NaBF4, NaH2PO4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF6- > ClO4- approximately BF4- > H2PO4-. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.

Effect of the counter-anion type and concentration on the liquid chromatography retention of beta-blockers.

Analysis of beta-blockers, basic pharmaceutical compounds with pKa values greater than 8.5, in reversed-phase HPLC can sometimes be challenging in terms of selection of the mobile phase pH, buffer concentration, and acidic modifier. The effect of the type and concentration of various mobile phase additives on the reversed-phase HPLC retention of these compounds was studied. HPLC analysis was performed at a mobile phase pH of 3 ensuring the protonation of the beta-blockers. It was found that at increasing perchlorate anion concentration at a constant mobile phase pH the retention factor for all beta-blocker compounds studied increased to varying degrees. The relative increase in the retention was attributed to ion interaction with the anionic mobile phase additive. Similar trends were observed when other types of inorganic salts such as NaH2PO4, NaPF6, NaBF4, and CF3CO2Na were employed. Differences in selectivity of the beta-blockers were obtained at a constant pH and an equimolar concentration of the different additives throughout the whole concentration range studied.

Concentration determination of methyl magnesium chloride and other Grignard reagents by potentiometric titration with in-line characterization of reaction species by FTIR spectroscopy.

A potentiometric titration method for methyl magnesium chloride and other Grignard reagents based on the reaction with 2-butanol in THF has been developed and validated. The method employs a commercially available platinum electrode, using an electrolyte compatible with non-aqueous solvents. Well-defined titration curves were obtained, along with excellent method precision. The endpoint was precisely determined based on the first derivative of the titration curve. Different solvents such as THF, diethyl ether and methylene chloride provided similar results with regard to sharpness of the endpoint and method precision. The method was applied to a wide array of Grignard reagents including methyl magnesium bromide, ethyl magnesium chloride, propyl magnesium chloride, vinyl magnesium chloride, phenyl magnesium chloride, and benzyl magnesium chloride with similar precision and accuracy. Application of in-line FTIR was demonstrated for in situ monitoring of the titration reaction, allowing characterization of the reaction species. An authentic spectrum of the MeMgCl-THF complex was obtained using spectral subtraction and the vibrational absorbance bands were identified. FTIR also provided an alternative for detecting the titration endpoint, and the titration results so obtained, provided a cross-validation of the accuracy of the potentiometric titration.