Feasibility of supercritical fluid chromatography/mass spectrometry of polypeptides with up to 40-mers.

Supercritical fluid chromatography (SFC) provides a number of advantages over traditional HPLC such as speed, practical use of longer columns, a normal-phase retention mechanism, and reduced use of organic solvents. Yet, it has been a technique traditionally limited to relatively nonpolar compounds. The nature of SFC mobile and stationary phases did not allow the elution of ionic compounds or of peptides, except, in the latter case, for the most hydrophobic peptides. The characterization of peptides is critically important for drug discovery and development in the pharmaceutical industry, as well as for a variety of other important applications. Here, for the first time to our knowledge, we show that relatively large peptides (at least 40 mers), containing a variety of acidic and basic residues, can be eluted in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column, which was specifically developed for SFC, was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric detection.

Automated metabolic profiling analysis of urinary steroids by a gas chromatography mass spectrometry data system.

A computer system (MSSMET), using methylene unit retention indices for an off-line reverse library search analysis of selected ion chromatograms from gas-liquid chromatographic mass spectrometric data, has been applied for the qualitative and quantitative determination of daily variations in the excreted levels of urinary steroids of two individuals, using capillary column gas-liquid chromatography. Aliquots of 24 h urine collections and morning spot urine samples were examined. The daily excretion pattern of most of the major steroid metabolites was fairly consistent from day to day (i.e. 3 alpha-hydroxy-5 alpha-androstane-17-one, androsterone; 3 alpha-hydroxy-5 beta-androstane-17-one, etiocheolanolone; 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnane-11,20-dione, THE; 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 beta-pregnane-20-one, THF; 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 alpha-pregnane-20-one; allo-THF; 3 alpha, 17 alpha, 20 alpha, 21-tetrahydroxy-5 beta-pregnane-11-one, cortolone; 3 alpha, 17 alpha, 20 beta, 21-tetrahydroxy-5 beta-pregnane-11-one, beta-cortolone; 5 beta-pregnane-3 alpha, 11 beta,17 alpha,20 alpha,21-pentol, cortol; 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20 beta, 21-pentol, beta-cortol), while certain other steroid metabolites had a less consistent excretion pattern (3 beta-hydroxy-5-androstene-17-one, for example). Advantages and disadvantages of using capillary columns for the automated metabolic profile analysis of urinary steroids by reverse library search of selected mass chromatograms.

Combined supercritical fluid extraction/solid-phase extraction with octadecylsilane cartridges as a sample preparation technique for the ultratrace analysis of a drug metabolite in plasma.

Supercritical fluid extraction was coupled with solid-phase extraction using octadecylsilane cartridges for the selective isolation of ultratrace levels of a drug metabolite, mebeverine alcohol, from plasma. Plasma was directly applied to the extraction cartridge, the cartridge was washed to remove protein and then extracted under supercritical conditions using CO2/5% methanol. The effluent from the extraction cell was bubbled through a small volume of 2-propanol to trap the extracted mebeverine alcohol. The effects of extraction pressure and temperature on analyte recovery were examined. The absolute recovery, selectivity, precision, and accuracy of the combined supercritical fluid extraction/solid-phase extraction approach were compared to those of conventional solid-phase extraction using gas chromatography/mass spectrometry in the selected-ion monitoring mode. Mebeverine alcohol was used as a model compound, and dog plasma was employed as the biological matrix for these studies.

Evaluation of capillary supercritical fluid chromatography with mass spectrometric detection for the analysis of a drug (mebeverine) in a dog plasma matrix.

Supercritical fluid chromatography with mass spectrometric detection was evaluated as a technique for the analysis of drugs in biological fluids. Dog plasma was spiked with a model drug, mebeverine, and with a deuterium-labeled analog of mebeverine. The spiked plasma was prepared for analysis by solid-phase extraction on octadecylsilane cartridges. Mebeverine levels in the spiked dog plasma samples were determined by interpolation from a standard curve. Accuracy and precision of the analysis were determined within and between days. In general, accuracy was found to be 100 +/- 15% for plasma samples spiked with 6 to 60 ng mebeverine/ml. The relative standard deviation for replicate sample analysis over this concentration range was between 5 and 12.5%.

Increasing bioanalytical throughput using pcSFC-MS/MS: 10 minutes per 96-well plate.

The utility of packed-column supercritical, subcritical, and enhanced fluidity liquid chromatographies (pcSFC) for high-throughput applications has increased during the past few years. In contrast to traditional reversed-phase liquid chromatography, the addition of a volatile component to the mobile phase, such as CO2, produces a lower mobile-phase viscosity. This allows the use of higher flow rates which can translate into faster analysis times. In addition, the resulting mobile phase is considerably more volatile than the aqueous-based mobile phases that are typically used with LC-MS, allowing the entire effluent to be directed into the MS interface. High-throughput bioanalytical quantitation using pcSFC-MS/MS for pharmacokinetics applications is demonstrated in this report using dextromethorphan as a model compound. Plasma samples were prepared by automated liquid/liquid extraction in the 96-well format prior to pcSFC-MS/MS analysis. Three days of validation data are provided along with study sample data from a patient dosed with commercially available Vicks 44. Using pcSFC and MS/MS, dextromethorphan was quantified in 96-well plates at a rate of approximately 10 min/plate with average intraday accuracy of 9% or better. Daily relative standard deviations (RSDs) were less than 10% for the 2.21 and 14.8 ng/mL quality control (QC) samples, while the RSDs were less than 15% at the 0.554 ng/mL QC level.

Comparison of packed-column supercritical fluid chromatography--tandem mass spectrometry with liquid chromatography--tandem mass spectrometry for bioanalytical determination of (R)- and (S)-ketoprofen in human plasma following automated 96-well solid-phase extraction.

The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies (pcSFC) for enantiomeric separations has increased steadily over the past few years. The addition of a significant amount (typically 20-95%) of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations. For example, higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher in pcSFC. Ultratrace enantioselective quantitation of drugs in biomatrixes is an ideal application for these chromatographic attributes. To demonstrate the utility of this approach, a pcSFC tandem mass spectrometry (pcSFC-MS/MS) method was compared to a LC-MS/MS method for quantitation of the (R)- and (S)-enantiomers of ketoprofen (kt), a potent nonsteroidal, anti-inflammatory drug, in human plasma. After preparation using automated solid-phase extraction in the 96-well format, kt enantiomers were separated on a Chirex 3005 analytical column using isocratic conditions. Validation data and study sample data from patients dosed with either orally or topically administered ketoprofen were generated using both pcSFC and LC as the chromatographic methods to compare and contrast these analytical approaches. Generally, most analytical attributes, including specificity, linearity, sensitivity, accuracy, precision, and ruggedness, for both of these methods were comparable with the exception that the pcSFC separation provided a roughly 3-fold reduction in analysis time. A 2.3-min pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, demonstrating a significant time savings for analysis of large batch pharmacokinetic samples using pcSFC.