Rosario LoBrutto

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.

Liophilic Mobile Phase Additives in Reversed Phase HPLC

Abstract The separation of basic compounds can be challenging and the use of inorganic mobile phase additives have been successfully used in chromatographic methods development. About fifteen years ago the role of these additives as ion-interaction agents for selectively adjusting retention of ionic analytes was discovered and the theory of chaotropicity was applied to reversed phase chromatography. In the last ten years the studies of the influence of these counterions on the retention of ionizable analytes and the interaction with the stationary phase in various hydro-organic eluents has expanded our knowledge of this phenomenon. The general view and understanding of the process have been significantly updated and the use of these ionic additives (liophilic ions) in the mobile phase has become regular practice in the pharmaceutical industry for optimization and fine tuning of complex separations. This paper reviews the latest developments in the field and discusses the modification and expansion of our theoretical understanding of the process. The paper also describes their application in practical separations for a wide variety of analytes, from small molecules to peptides and even chiral separations.

INVESTIGATION OF THE EFFECT OF PRESSURE AND LIOPHILIC MOBILE PHASE ADDITIVES ON RETENTION OF SMALL MOLECULES AND PROTEINS USING REVERSED-PHASE ULTRAHIGH PRESSURE LIQUID CHROMATOGRAPHY

The goal of this study was to investigate the effect of pressure modulation on the retention, selectivity, and peak efficiency of proteins and small basic and neutral molecules using reversed-phase liquid chromatography. A backpressure restrictor was used in the experiments to study pressure variation at a constant flow rate. Experiments were conducted in isocratic mode at constant temperature using mobile phase additives namely sodium perchlorate and potassium hexafluorophosphate. Upon increase of pressure at constant flow rate, an increase of the retention factor of analytes was observed depending on the type of analyte and molecular weight of the compound. However, retention factors of all analytes decreased with an increase in flow rate at constant pressure. Pressure alone can have a significant effect on retention of polar and ionized analytes; however, the effects are much more pronounced with larger analytes such as proteins in the presence of liophilic mobile phase additives. Selectivity changes could be obtained for a mixture of analytes by modulating the level of backpressure applied and this could be an effective tool in HPLC method development. The effects of pressure should be considered when transferring methods from conventional HPLC applications to fast LC applications at high pressures, especially for proteins and large peptides. In the case of ionized large molecules and proteins, strong liophilic additives are recommended for use in the mobile phase to streamline the transfer of a method originally developed on a fast LC system to a conventional LC system or vice versa.

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.

Enhancing Productivity in the Analytical Laboratory Through the Use of Ultra Fast-HPLC in Preformulation/Formulation Development

Abstract The pharmaceutical industry today is driven to create new, more efficient ways to discover, develop, deliver and monitor drugs. Pharmaceutical companies are being faced with major challenges in reducing drug discovery and development timelines. Automation and the introduction of new analytical technologies that increase speed of analysis are integral in the analytical laboratory. The development of rapid chromatographic methods in preformulation and formulation development is playing an increasing role to support this drive in efficiency and productivity. The introduction of ultra fast-HPLC systems that can operate at pressures of up to 15,000 psi with columns packed with sub-2-µm particles have allowed for high speed and efficient separations. The consequent reduction of time, solvent, and waste disposal, and the analysis of more samples per unit time makes ultra-fast HPLC a very attractive technology. Faster method development and decision making can be achieved during late-phase preformulation/formulation development for the analysis of both singly charged and multiply charged basic compound analysis. The use of ultra fast-HPLC (UHPLC) for pH scouting experiments and the determination of the analytes ionogenic nature was shown to be an effective tool for rapid and systematic method development. The implementation of this technology was also evaluated for the analysis of drug product formulations and excipient compatibility studies. Increased speed of analysis and significant gains in resolution per unit time were obtained compared to separations performed using conventional HPLC systems (operating pressures of <5,500 psi). Also, the use of liophilic ions as mobile phase additives with operation under high pressures led to enhanced separation selectivity, retention, and peak symmetry of multiply charged basic compounds.

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.

Kinetic analysis and subambient temperature on-line on-column derivatization of an active aldehyde

The chromatographic analysis of aldehydes under typical reversed-phase conditions may be a challenging task due to an equilibrium process leading to the formation of a gem diol species regardless of acidic or basic conditions. Initially, a reversed-phase HPLC gradient elution was developed to determine the amount of a n acetylenic aldehyde in a reaction mixture. Significant fronting was observed under acidic and basic conditions even at -5 degrees C. In order to circumvent this problem, a reversed-phase HPLC gradient method on a C18 column at subambient temperature was developed using diethylamine as a mobile phase additive for on-line on-column derivatization of the aldehyde moiety. The on-line on-column reaction rate for the derivatization of the aldehyde with diethylamine was determined as a function of column temperature. An Arrhenius plot was constructed and the activation energy was calculated. The chromatographic behavior of the derivatized acetylenic aldehyde and products formed in-situ in the chromatographic system were studied at various temperatures ranging from -10 to 60 degrees C. It was found that the reaction products could be controlled by adjusting the column temperature. Different reaction pathways were identified as a function of temperature. The products and the reaction pathways were characterized by NMR, LC-MS and UV spectra.

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.