Enantiomeric purity analysis of synthetic peptide therapeutics by direct chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, the undesirable d-isomers can be introduced as impurities in amino acid starting materials and can also be formed during peptide synthesis and in some cases during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity of a peptide. The peptide is hydrolyzed in deuterated acid to facilitate correction for any racemization occurring during this step of sample preparation, and the amino acids are subsequently separated by chiral chromatography interfaced with ESI-MS/MS for quantitation. The amino acid samples are analyzed directly following hydrolysis using high-low chromatography and extraction of selected ion response, providing efficiency and simplicity by avoiding the derivatization steps and multiple external standards required by traditional methodologies. GMP method validation feasibility is described for all nineteen chiral proteogenic amino acids. The practical application of the chiral HPLC-ESI-MS/MS method was demonstrated through the recovery of d-amino acid substitutions at each residue of an octapeptide across the 0.1-1.0 % range of interest. The method was applied to the analysis of four model peptides, each consisting of 8-14 amino acid residues, and the results were comparable to those provided by traditional testing methods.

Evaluation of a hydrophilic interaction liquid chromatography design space for sugars and sugar alcohols.

Based on a column-screening exercise, a column ranking system was developed for sample mixtures containing any combination of 26 sugar and sugar alcohol analytes using 16 polar stationary phases in the HILIC mode with acetonitrile/water or acetone/water mobile phases. Each analyte was evaluated on the HILIC columns with gradient elution and the subsequent chromatography data was compiled into a statistical software package where any subset of the analytes can be selected and the columns are then ranked by the greatest separation. Since these analytes lack chromophores, aerosol-based detectors, including an evaporative light scattering detector (ELSD) and a charged aerosol detector (CAD) were employed for qualitative and quantitative detection. Example qualitative applications are provided to illustrate the practicality and efficiency of this HILIC column ranking. Furthermore, the design-space approach was used as a starting point for a quantitative method for the trace analysis of glucose in trehalose samples in a complex matrix. Knowledge gained from evaluating the design-space led to rapid development of a capable method as demonstrated through validation of the following parameters: specificity, accuracy, precision, linearity, limit of quantitation, limit of detection, and range.

Aerosol-based detectors for liquid chromatography.

Aerosol-based detectors developed within the last few decades have increasingly addressed the need for sensitive, universal liquid chromatography detection in a wide variety of applications. Herein, we review the operating principles, instrumentation, analytical characteristics, and recent applications of the three general types of such detectors: evaporative light scattering detection (ELSD), condensation nucleation light scattering detection (CNLSD); commercially known as the nano-quantity analyte detector (NQAD), and charged aerosol detection (CAD). Included is a comparative evaluation of the operational and analytical characteristics of these detectors.

Analysis of magnesium from magnesium stearate in pharmaceutical tablet formulations using hydrophilic interaction liquid chromatography with nano quantity analyte detection.

This study demonstrates the use of hydrophilic interaction liquid chromatography with a nano quantity analyte detector for the retention, separation and detection of magnesium from magnesium stearate in tablet formulations for a drug product formulation blend containing a hydrochloride salt of a weakly basic compound as the active ingredient. The nano quantity analyte detector can provide direct detection of inactive excipients and inorganic salts lacking ultraviolet chromophores, as well as, all non-volatile compounds. The separation was accomplished using a SeQuant ZIC-HILIC column and mobile phase consisting of 32.5:32.5:35 of acetone/methanol/ammonium formate buffer (150 mM, pH 4.5). Common validation parameters were evaluated to assess the method's quantitative potential for magnesium (from magnesium stearate) including: linearity, accuracy, specificity, solution stability, repeatability, and intermediate precision. Overall, the method described in this report proved to be very robust and represents a novel technique to conveniently separate and detect magnesium from magnesium stearate in pharmaceutical preparations both quickly and accurately.

Hydrophilic interaction chromatography with aerosol-based detectors (ELSD, CAD, NQAD) for polar compounds lacking a UV chromophore in an intravenous formulation.

In this work, a high performance liquid chromatography (HPLC) method is reported for the separation and quantitation of a drug substance that is highly polar and lacking a chromophore in a mannitol intravenous (IV) formulation. Three polar stationary phases operated in hydrophilic interaction chromatography (HILIC) mode were evaluated in conjunction with an Alltech 800 ELSD detector. These columns were evaluated with respect to chromatographic properties such as buffer, pH and organic concentrations to identify the best stationary phase. The chromatographic method was then validated for the determination of mGlu2/3 receptor agonist (-)-(1R, 4S, 5S, 6S)-4-Amino-2-sulfonylbicyclo [3.1.0] hexane-4,6-dicarboxylic acid (LY404039) content in a mannitol IV formulation with respect to linearity (R(2) of 0.9997), repeatability (%RSD of 0.36%), accuracy, solution stability (99.56% after 24h), specificity, intra-assay precision (%RSD 0.48%) and limit of detection (LOD, ∼50 µg/mL). In addition to the Alltech 800 ELSD detector, several other aerosol-based detectors were investigated for reproducibility, linearity and LOD. These additional detectors consisted of an Alltech 3300 evaporative light scattering detector (ELSD), a nano quantity analyte detector (NQAD) and a charged aerosol detector (CAD). Based on the data from this report, a feasible isocratic LC method was achieved using a TSKgel Amide-80 column with mobile phase conditions of 30% water with 0.2% trifluoroacetic acid (TFA) and 70% acetonitrile (ACN) using any of the four aerosol-based detectors for detection and quantitation.

Weak bases and formation of a less soluble lauryl sulfate salt/complex in sodium lauryl sulfate (SLS) containing media.

This work reports on the solubility of two weakly basic model compounds in media containing sodium lauryl sulfate (SLS). Results clearly show that the presence of SLS in the media (e.g. simulated gastric fluid or dissolution media) can result in an underestimation of solubility of some weak bases. We systematically study this phenomenon and provide evidence (chromatography and pXRD) for the first time that the decrease in solubility is likely due to formation of a less soluble salt/complex between the protonated form of the weak base and lauryl sulfate anion.

A simple and efficient approach to reversed-phase HPLC method screening.

The development and utility of an efficient HPLC method screening strategy using only four columns for the separation of pharmaceutical compounds and related impurities is presented. The strategy established a two-column approach to enable rapid early method development, along with a four-column approach for commercial method development of the analytical methods utilized to verify the quality of drug substance or drug product. Mobile phases consisted of acetonitrile or methanol with aqueous trifluoroacetic acid for low pH screening, and ammonium hydroxide for high pH screening. Examples are provided to demonstrate the practicality and orthogonality of the method screening process. A unique system suitability check, using commercially available compounds, was incorporated as a tool for troubleshooting and for ensuring adequate system performance prior to screening. Initial testing of the strategy revealed that the columns chosen were successful in leading to assay and impurity methods for 40 pharmaceutical compounds.

A critical evaluation of fasted state simulating gastric fluid (FaSSGF) that contains sodium lauryl sulfate and proposal of a modified recipe.

The aim of this work is to evaluate one of the most commonly used fasted state simulating gastric fluids (FaSSGFs), which contains sodium lauryl sulfate (SLS) (FaSSGF(SLS)), and propose a more appropriate surfactant concentration. Surface tension studies clearly show that the critical micelle concentration (CMC) of SLS in the relevant media (a media whose pH and sodium chloride concentration are representative of physiological conditions) is significantly lower (p<0.05) than 8.67 mM, which is the SLS concentration in FaSSGF(SLS). The CMC of SLS in the relevant media was determined to be 1.75 mM. Based on this a modified recipe is proposed in which the concentration of SLS is sufficient to achieve a surface tension similar to that in vivo without causing artificial micellar solubilization. Solubility, intrinsic dissolution, and GastroPlus modeling studies are presented to support and give rationale for the modified recipe. In addition, a comparison between the modified recipe and other FaSSGFs reported in the literature is made.

Empirical observations and mechanistic insights on the first boron-containing chiral selector for LC and supercritical fluid chromatography.

Boromycin is a macrodiolide that exists as a hydrophobic Böeseken complex formed from boric acid and a chiral polyhydroxy macrocyclic ligand. It was covalently bonded to silica gel through a urea linkage to an attached d-valine ester. When evaluated as a chiral stationary phase, it shows pronounced enantioselectivity toward primary amine-containing racemates, separating 98% of those tested. The selectivity is most pronounced in the presence of organic solvents and supercritical CO2 + methanol. The enantioselective binding site and mechanistic factors are examined. Analytes can be complexed as either the free base or their protonated analogues, with the free base being more strongly associated with the chiral selector.

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems.

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.

Analysis of mannitol in pharmaceutical formulations using hydrophilic interaction liquid chromatography with evaporative light-scattering detection.

This study demonstrates the use of hydrophilic interaction liquid chromatography (HILIC) for the separation of both active and inactive ingredients in pharmaceuticals from a single injection. Excipients commonly used in parenteral formulations were separated using a gradient method employing increasing aqueous composition. An evaporative light-scattering detector (ELSD) provided direct detection of inactive excipients and inorganic salts lacking UV chromophores. Analyses of Gemzar parenteral formulations using optimized isocratic HILIC-ELSD method conditions were performed based on retention time screening from the gradient assay. All of the components were efficiently separated using a TSK-Gel Amide 80 column including gemcitabine, mannitol, and sodium cation demonstrating the qualitative capability of the technique. The method was thoroughly validated for mannitol content to access the quantitative potential of the technique. Validation parameters included linearity, accuracy, specificity, solution stability, repeatability, and intermediate precision. Overall, the method described in this report proved to be very robust and represents a novel technique to conveniently separate and detect the active and inactive components in pharmaceuticals both quickly and accurately.

Evaluation of a monolithic silica column operated in the hydrophilic interaction chromatography mode with evaporative light scattering detection for the separation and detection of counter-ions.

In this work a monolithic silica column operated in the hydrophilic interaction chromatography (HILIC) mode in conjunction with an evaporative light scattering detector (ELSD) was investigated. Lithium, sodium and potassium were used as the test counter-ions for this evaluation. Chromatographic properties of this column operated in the HILIC mode were determined by varying key mobile phase parameters, such as pH, flow rate, buffer strength, acid and organic modifier. As organic content was increased from 60 to 90% acetonitrile, retention time increased on average by a factor of seven for the test cations listed above. Buffer concentration and pH were also observed to have an effect, although not as significant as the HILIC effect that was observed by changing organic content. Flow rates up to 5 mL/min were utilized to perform counter-ion separations in less than 3 min. After examining the changes in retention, resolution, and peak shape an optimized method was established and then further evaluated for linearity, reproducibility, and limit of detection (LOD) for sodium. Linearity was acceptable with an R2 value of 0.999 across the working-standard range and a LOD of 0.1 microg/mL was calculated. The reproducibility on the counter-ion determination from pharmaceutical sodium salts was 1.6% R.S.D. on average, and the accuracy of the counter-ion prediction was approximately 3% from theory when salt content was corrected for potency.

Enantiomeric separation of dansyl amino acids using macrocyclic antibiotics as chiral mobile phase additives by narrow-bore high-performance liquid chromatography.

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.