Analysis of conjugated bile acids by on-line supercritical fluid chromatography/thermospra mass spectrometry

A rapid method was developed for the analysis or polar conjugated bile acids by combined packed column super-critical fluid chromatography/mass spectrometry. Direct coupling of the column to the mass spectrometer was effected with a modified thermospray interface operated in the filament-on mode. Maximum sensitivity for the bile acid conjugates was achieved by recording the negative ions generated in the ionization process. The effects of vaporizer and source temperatures, repeller voltage and discharge conditions on the bile acid response were investigated. The mass spectra obtained for the common glycine and taurine conjugates yielded only the [M – H]^? pseudo-molecular ions. In contrast with the taurine derivatives, the glycine forms produced a weak ion current and exhibited broad hands. The applicability of the technique is illustrated with a sample of human bile.     

Clinical and pharmaceutical applications of packed-column supercritical fluid chromatography

Packed-column supercritical fluid chromatography (pSFC) is a fast separation technique that combines the properties of HPLC and GC. pSFC with carbon dioxide as the mobile phase and packed silica column as the stationary phase possesses the properties of normal phase mechanism; however, the addition of modifiers to the mobile phase allows the separation of relatively polar compounds. In spite of its many positive attributes, pSFC has not been widely used in areas such as proteomics, where methods such as HPLC dominate. Packed column SFC has been extensively used in clinical and pharmaceutical laboratories, especially for separation of nonpolar and chiral drugs. This review will discuss recently published applications of pSFC, with a specific focus on its advantages and limitations for the analysis of pharmaceuticals with varying chemical properties.     

Mass-directed fractionation and isolation of pharmaceutical compounds by packed-column supercritical fluid chromatography/mass spectrometry.

An automated packed-column semi-preparative supercritical fluid chromatography/mass spectrometry (SFC/MS) system incorporating mass-directed fraction collection has been designed and implemented as an alternative to preparative HPLC and preparative HPLC/MS (PrepLC/MS) for the purification of pharmaceutical compounds. The system incorporates a single quadrupole mass spectrometer and a supercritical fluid chromatograph. Separations were achieved using a binary solvent system consisting of carbon dioxide and methanol. Purification of SFC-separated compounds was achieved incorporating mass-directed fraction collection, enabling selective isolation of the target molecular weight compound and eliminating the collection of undesired compounds (e.g., by-products, excess starting materials, etc.). Cross contamination between fractions and recoveries of the system were investigated. Mass spectrometer ionization with basic mobile additives is discussed, and examples of preparative SFC/MS chiral separations are presented. Early experiences suggest SFC will be a powerful and complementary technique to HPLC for the purification of pharmaceutical compounds.     

Glycolipid class profiling by packed-column subcritical fluid chromatography

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.     

Development and application of packed-column supercritical fluid chromatography/pneumatically assisted electrospray mass spectrometry

A pneumatically assisted electrospray liquid chromatography/mass spectrometry (LC/MS) interface has been modified for use with packed-column supercritical fluid chromatography (pcSFC). The modifications include the addition of a concentric sheath-flow liquid to the spray device. This allowed the addition of modifiers at the sprayer tip that promote ionization of neutral, pcSFC-separated components. Post-column chromatographic fidelity was preserved using a novel pressure-regulation scheme. Post-column pressure regulation was accomplished by adding a “pressure-regulating fluid” (supplied under pressure control) to the effluent just ahead of the sprayer. The modified interface has been used to characterize a variety of mixtures including emollients, modified polysiloxanes, and pharmaceutical agents. The spectra produced by using this pcSFC/MS interface are similar to electrospray LC/MS spectra.     

Analysis of artemisinin by a packed-column supercritical fluid chromatography-atmospheric pressure chemical ionisation mass spectrometry technique

A packed-column supercritical fluid chromatography-atmospheric pressure chemical ionisation mass spectrometry method was studied for the determination of artemisinin from Artemisia annua L. extracts. The technique does not require any kind of derivatisation prior to the analysis. All samples were simply dissolved in methanol and injected into the mobile phase. Detection was achieved by using mass spectrometry with atmospheric pressure chemical ionisation. The ionisation technique is relatively soft and provides protonated molecular ion and informative structural fragmentation for the compound. Benzophenone was used as a chromatographic standard for the determination of the analytical reproducibility. The supercritical carbon dioxide mobile phase used in the system was modified by 10% methanol. The average absolute retention time was 3.54 min with a standard deviation of 0.017 min and a relative standard deviation of 0.4% with respect to benzophenone for the procedure. The correlation coefficient was 0.998 and detection limit 370 pg on column.     

The analysis of beta-agonists by packed-column supercritical fluid chromatography with ultra-violet and atmospheric pressure chemical ionisation mass spectrometric detection

Packed-column supercritical fluid chromatography (pSFC) using ultra-violet (UV) and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS) provides a versatile method for the identification and quantification of beta-agonists. We have achieved good separation of clenbuterol, salbutamol, terbutaline and fenoterol with good resolution and reasonable retention times using a high concentration of methanol modifier in the supercritical CO2, together with small amounts of both acidic (trifluoroacetic acid, TFAA) and basic (triethylamine, TEA, or diethylamine, DEA) additives. APCI-MS gave unambiguous identification of the 4 analytes, and increasing cone voltage provided informative fragmentation patterns. The pSFC-MS technique was shown to be linear (R2 > or = 0.996) over the concentration range 1-50 micrograms ml-1. Single ion monitoring (SIM) gave detection limits (on-column) of 2.5 ng (clenbuterol), 0.83 ng (terbutaline), 7.6 ng (salbutamol) and 2.7 ng (fenoterol). The pSFC-MS system was shown to be reproducible within a day, between days, and between restrictors. Analysis of milk samples 'spiked' with beta-agonists showed that the matrix caused no interference, with detection limits of approximately 500 micrograms l-1 of beta-agonists. More dilute solutions could be analysed by pre-concentration before the SFC stage.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Analysis of sulphonamides using supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry

Packed-column supercritical fluid chromatography has been used for the separation of mixtures of sulphonamides on silica and amino-bonded stationary phases utilizing carbon dioxide with methanol modifier as the mobile phase. The effect of modifier concentration, column pressure and modifier identity on retention was also studied. Packed-column supercritical fluid chromatography-mass spectrometry (SFC-MS) of these mixtures utilizing both moving-belt and modified thermospray interfaces was also studied. The identification of sulphamethazine in a spiked porcine kidney extract was performed by SFC-MS using the moving-belt interface.     

Analysis of commercial dyes by capillary column supercritical fluid chromatography

High resolution separations of selected commercial azo, aniline, and anthraquinone dyes by capillary column supercritical fluid chromatography (SFC) are demonstrated. Supercritical n-pentane was used as a mobile phase and provided efficient separations of multi-functional, polar disperse dyes with molecular weights up to approximately 700 daltons.     

The retention behaviour of conjugated bile acids in reversed phase high performance liquid chromatography.

The retention behaviour of conjugated bile acids has been studied in a reversed phase high performance liquid chromatographic (RP-HPLC) system by using the mixture of methanol and aqueous phosphate buffer as the mobile phase. The retentions of the conjugates in RP-HPLC have been found to be mainly controlled by the glycine and taurine groups. The selectivity between five different glycine and taurine conjugated bile acids is a constant in RP-HPLC. This selectivity has been used for peak identification in the practical separation of conjugated bile acids.     

Analysis of various classes of drugs by capillary supercritical fluid chromatography

Three classes of drugs were screened for analysis feasibility by capillary column supercritical fluid chromatography. These included steroids, therapeutic antibiotic drugs, and drugs of abuse, such as cannabinoids. Supercritical fluid carbon dioxide was used as the mobile phase in conjunction with a methylpolysiloxane stationary phase capillary column and a flame ionization detector. All compounds considered were analyzed either as single component solutions, simple mixtures, or in actual complex mixtures.     

Determination of diagnostically important free and conjugated bile acids in blood plasma by high-performance liquid chromatography

A procedure was proposed for the determination of free bile acids and their conjugates in blood plasma by the reversed-phase HPLC using the column Lichrospher 100 RP-18 (250 + 4.6 mm) with gradient elution and UV-detection at 206 nm. The procedure allowed the simultaneous determination of diagnostically important cholic acids, tauro-and glyco-cholates in blood plasma of patients with no preliminary separation of the analytes into subtypes. The bile acids and their conjugates were isolated from the sample matrix by solid phase extraction in a Sep-Pack C18 cartridge. The limits of detection were 0.11–0.15 mM for free acids and 0.015–0.025 mM for conjugates.     

Enantiomeric resolution of bifonazole by supercritical fluid chromatography

The enantiomeric separation of bifonazole by supercritical fluid chromatography on Chiralpak AD has been studied. The effect of different modifiers (methanol, ethanol, 2-propanol, and acetonitrile) was examined. Enantioseparation was possible with all of them, but the best results were provided by the alcohol-type ones. The resolution was higher than 5 in all cases. The isoelution temperatures Tiso were calculated from the study of the temperature effect for the different organic modifiers. The value of Tiso was below the working temperature range on using methanol, but above it with ethanol or 2-propanol.     

Determination of thermodynamic properties by supercritical fluid chromatography

This survey attempts to summarise thermodynamic applications of supercritical fluid chromatography (SFC) with an emphasis on the results published during the last 10 years. In addition to a review of thermodynamic measurements by SFC, it contains brief sections on instrumental considerations and on the sources of auxiliary information needed when processing the retention data.     

Trace analysis of agrochemicals by supercritical fluid chromatography

Capillary supercritical fluid chromatography was performed using solvent-vented injection in conjunction with a thermionic ionization detector. Mixtures of organo-nitrogen and phosphorus containing agrochemicals were chromatographed and detection of low picogram quantities was observed.     

High-resolution analysis of polyprenols by supercritical fluid chromatography

A high-resolution analysis of polyprenol mixtures was achieved by supercritical fluid chromatography (SFC). The separation of polyprenols was examined on an octadecylsilane-packed column with liquid carbon dioxide as the mobile phase and ethanol as modifier. Using this chromatography system, the resolution of separation (Rs) between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) was two times higher than that using conventional reversed-phase high-performance liquid chromatography. Our SFC technique allows the advantage of baseline separation of polyprenol samples containing hydrophobic components such as terpenes or fatty acids that are unfavorable for good separation. This method is very useful for the analysis of structurally close polyprenol analogues of rubber plant metabolites.