Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Characterization of ouabain-like immunoreactivity in human urine.

To characterize ouabain-like immunoreactivity in human urine. Sensitive radioimmunoassay for ouabain characterized by high-performance liquid chromatography. Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86 nmol (62.5 micrograms) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7 nmol/l 8 min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain. A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma.

One-step immunoaffinity purification and partial characterization of hypophyseal growth hormone from the African catfish, Clarias gariepinus (Burchell)

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T3 plasma levels in juvenile catfish.

Urotensin II in the central nervous system of the frog Rana ridibunda: immunohistochemical localization and biochemical characterization.

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians.

Antiproteases attenuate the release of neutrophil chemotactic activity from bronchial epithelial cells induced by smoke.

The released neutrophil chemotactic activity (NCA) from bronchial epithelial cells (BECs) in response to smoke extract was evaluated by reverse-phase, high-performance liquid chromatography (RP-HPLC) and the involvement of proteolytic activity was assessed for the release of NCA from BECs. Smoke extract stimulated the release of NCA (55.3 +/- 5.2 vs. 17.3 +/- 4.1 cells per high-power field [HPF], p < .001). The released activity determined by RP-HPLC analysis was 15-hydroxyeicosatetraenoic acid and leukotriene B4. Several structurally and functionally different serine protease inhibitors, including alpha-1-protease inhibitor (alpha-1-PI), chloromethyl ketone (CK) derivatives, N-tosyl-L-lysine CK (TLCK), methoxysuccinyl-Ala-Ala-Pro-Val CK (SPCK), N-alpha-tosyl-L-phenylalanine CK (TPCK), and N-alpha-p-tosyl-L-arginine methyl ester hydrochloride (TAME), attenuated the release of NCA (P < .01) in a dose-dependent fashion. Leupeptin, a cysteine protease inhibitor, has only a small effect on the release of NCA (p < .05), and phosphoramidon, a neutral endopeptidase inhibitor, had no effect. The measurement of proteolytic enzyme activity using synthetic substrate S-2288 revealed that smoke extract significantly (p < .05) augmented the serine protease activity in BEC layers. Culture supernatant fluids and cell lysates of BECs in response to smoke extract solubilized 14C-labeled casein. These results suggest that BECs may release lipoxygenase-derived NCA in response to smoke extract and that the release of NCA may involve the activation of proteolytic activity of BECs which was inhibited by serine protease inhibitors.

Solid-phase synthesis of thymosin β10using a p-cyanotrityl resin. Chemical characterization and immunochemical control of the synthetic peptide

Thymosin β10(43 amino acids) was synthesized following the Fmoc-solid-phase strategy, using a new p-cyanotrityl resin. This resin shows enhanced acid stability compared with the usual trityl ester resins, thus providing selective peptide synthesis with high yield and purity. Crude Tβ10 was purified with gel filtration and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The purified synthetic peptide was shown to co-elute with recombinant Tβ10 in an analytical RP-HPLC system. The amino acid analysis data were in agreement with those reported for the literature primary structure of natural Tβ10. The electrospray ionization mass spectrometry (ESIMS) data showed almost identical average mass (Mr, exp)-values for the synthetic or the recombinant product, which were in agreement with the molecular mass calculated on the basis of the proposed primary structure for the peptide. Furthermore, the synthetic Tβ10 exhibited similar immunochemical characteristics compared with the recombinant material in an in vitro ELISA test using rabbit polyclonal anti-Tβ10 antiserum.

Antiprotease Activity in Urine of Patients with Inflammatory Skin Disorders

Polymorphonuclear leukocytes contain well-defined proteolytic enzymes in their azurophilic granules that can be released into tissues during inflammation, producing a localized excess of proteases that causes a protease-antiprotease imbalance with subsequent tissue destruction. The antiproteolytic compounds of the epidermis, such as the protease inhibitors elafin and antileukoprotease, are thought to counteract the proteolytic tissue damage. We investigated the urine of patients suffering from inflammatory skin conditions (e.g., erysipelas, psoriasis) for the presence of urinary antiprotease activities. Purification of elastase-inhibitory activities from pooled urine samples by cation exchange high-performance liquid chromatography and preparative and analytical reverse-phase high-performance liquid chromatography yielded two different types of inhibitors. One was a cationic, acid-stable, and elastase-specific inhibitor of M(r) 6,000 by size-exclusion high-performance liquid chromatography. N-terminal amino acid sequence analysis of the first 28 residues showed identity with elafin, an elastase-specific inhibitor recently isolated from psoriatic scales. The second anti-protease activity was due to two forms of urinary bikunin, the inhibitory subunit of inter-alpha-inhibitor. Both bikunin fragments, with M(r) 4,000 and 16,000, were identified by N-terminal amino acid sequence analysis of the first 10 residues and were characterized by an antiproteolytic profile against human leukocyte elastase, cathepsin G, and trypsin. Urinary protease inhibitors may serve as diagnostic markers of inflammatory diseases.

Identification of a new genetic variant of bovine beta-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis.

Various components of the beta-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants beta A1, beta A2, beta A3, and an unknown component previously denoted beta X [S. Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these components had relative molecular masses of 16 higher than the corresponding ones in the beta A1 map. The main differential peaks represented the 114-169 fragments of beta A1 and beta X, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145-156 sequence showed the 16 relative molecular mass difference. In beta X this sequence contained a Leu residue at position 152 instead of the Pro-152 in beta A1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence of the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named beta-casein F-5P.

Development of an analytical reversed-phase high-performance liquid chromatography assay for transforming growth factor β3

Transforming growth factor β3 (TGF-β3) is a dimeric protein ( M r 25 444) which regulates the proliferation of epithelial cells. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for TGF-β3 in order to investigate the solution stability of TGF-β3. Peak width and retention time were evaluated as a function of (1) the initial percent of acetonitrile, (2) the gradient slope, (3) the percentage of trifluoracetic acid, and (4) column temperature. By varying the above conditions, particularly temperature, it was possible to reduce retention times and peak widths by a factor of 2 and 6, respectively. The observed temperature effects on peak width were consistent with the concept that conformational changes can be induced in the protein during the chromatographic migration. A balance was found between on-column degradation and improved peak width at elevated temperatures. The final assay was found to be linear over a 10–150 μg/ml drug range with a correlation coefficient of 0.998 and acceptable reproducibility (R.S.D. 2.09%, n = 6). The assay has been useful for monitoring drug loss in ongoing stability studies; however, it was found to have poor resolving power when certain stressed samples of TGF-β3 were applied, suggesting that complimentary assay methodologies must be developed.

Solid-Phase Extraction Study and RP-HPLC Analysis of Lamotrigine in Human Biological Fluids and in Antiepileptic Tablet Formulations

Abstract An efficient off-line solid-phase extraction (SPE) of lamotrigine, 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine (LTG), a new antiepileptic drug, from human serum and urine, prior to high-performance liquid chromatographic analysis, was tested and optimized. High extraction recoveries were achieved from C8 Bond Elut cartridges (200mg/3ml), using acidic acetonitrile for the elution of LTG and the internal standard, 3,5-diamino-6-(2-methoxyphenyl)-1,2,4-triazine. Isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis on octylsilica, using a Lichrosorb RP-8, 5μm, 250x4.6 mm i.d. column and a mobile phase consisting of 0.05M acetate buffer pH5.6 and acetonitrile (72:28 v/v) proved to be sensitive and rapid. The identification of LTG was performed by UV detection at 306nm. The method detects approximately 0.9 ng of LTG on-column, using a 20-μl loop, and linearity holds from approximately 0.044 to 7.8 μg/ml in standard solutions. In plasma and urine, the limits of detec...

Modification of Chloride Ion Transport in Human Erythrocyte Ghost Membranes by Photoaffinity Labeling Reagents Based on the Structure of 5-Nitro-2-(3-Phenylpropylamino)-benzoic Acid (NPPB)

Using human red blood cell ghost membranes, we have evaluated 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid and 5-nitro-2-[N-3-(4-azido-2,3,5,6-tetrafluorophenyl)-propylamino]-benzoic acid (FAzNPPB) as photoaffinity labeling agents based on the structure of the widely important Cl− channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB). The tetrafluoro-substituted aryl azide was found to be a more effective photoinactivating agent than the corresponding protio compound. Using a tritiated version ([3H]FAzNPPB), we demonstrated that photoinactivation of Cl− flux was accompanied by photolabeling of the band 3 protein and membrane lipids. Both processes were diminished in the presence of NPPB and the related arylanthranilate flufenamic acid. Photolabeling resulted in the incorporation of 1.0 ± 0.2 mol 3H/mol protein in the band 3 integral membrane domain, whereas the cytoplasmic domain was essentially unlabeled, By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photolabeling was found to be the result of partial labeling of at least three different regions of the membrane domain, Based on trypsin proteolysis, reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry analysis, it is proposed that one of the sites of photolabeling is the peptide lys-phe-lys (590-592), FAzNPPB is a successful polyfluoro aryl azide photoaffinity labeling agent which may be of further use in studying the diverse effects of arylanthranilates on biological membranes.

Aerobic transformation of linear alkylbenzenesulphonates by mixed methane-utilizing bacteria

Biodegradation of commercial linear alkylbenzenesulphonate (LAS) was performed in shake flasks in the presence and absence of methane as a primary growth substrate for methanotrophs and as an inducer for catabolic enzymes. Methane-utilizing culture MM1 was dominated by type II methanotrophs and originated from a groundwater aquifer. Methane, carbon dioxide, and oxygen concentrations were measured by using gas chromatography. Disappearance of LAS, a mixture of C10-C13 homologues, was monitored by reversed-phase high-performance liquid chromatography (RP-HPLC). Methane reduced LAS transformation rate in a concentration dependent manner, suggesting competitive inhibition between natural substrate, methane, and fortuitous substrate, LAS. The fastest LAS transformation was determined in the absence of methane. Simultaneous methane oxidation and LAS degradation, and the inhibition of both transformation processes by acetylene, indicated the involvement of a methane-monooxygenase enzyme system in LAS transformation. RP-HPLC analysis showed that culture MM1 exhibited preferential capability to transform LAS with longer alkyl side-chain. Consequently, the highest transformation rate was observed with the homologue of C13 LAS.

High-temperature reversed-phase high-performance liquid chromatographic analysis of a synthetic copolymer on a non-porous support

High-temperature HPLC using a non-porous microparticulate reversed-phase column was evaluated for characterizing an exploratory magnetic resonance imaging polymeric contrast agent. High-efficiency separations were achieved allowing resolution of over 50 individual oligomers. Separations were optimized with respect to temperature and addition of neutral salt. Results were compared to those obtained with a traditional porous HPLC support as well as a macroporous perfusion-type column. Separation efficiencies were highest with the non-porous support followed in order by the macroporous and traditional porous packing. The utility of this technique for characterizing degradation of the polymer under stress conditions was demonstrated.

Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the α- or β-piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.

Biodegradation of undecylbenzenesulphonate by mixed methane-oxidizing culture

Biodegradation of undecylbenzenesulphonate (C 11LAS) was performed in shake flasks at 21°C by using a mixed methanotrophic-heterotrophic culture containing type II methanotrophs. Concentrations of C 11LAS and aromatic intermediates were determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Methane and carbon dioxide concentrations were measured in headspace samples by using gas chromatography. RP-HPLC analyses of aqueous samples show that the culture MM1 expresses the capability of C 11LAS transformation in the presence or absence of methane. Simultaneous methane oxidation and C 11LAS degradation, and the inhibition of both transformation processes by acetylene were observed. This suggests the possibility that C 11LAS transformation is catalyzed by methane monooxygenase (MMO). Comparable affinity of culture MM1 for both methane and C 11LAS ( K s = 4·7 and 5·7 μM, respectively), and more than four times higher maximum transformation rate for methane than for C 11LAS ( k = 0·42 and 0·096 mg mg −1 (dry weight) cells day −1, respectively), were determined. This further supports the involvement of MMO enzyme system in transformation and suggests a pronounced competitive inhibition of C 11LAS degradation by methane.

Reversed-Phase High-Performance Liquid-Chromatographic Behavior of Phthalic Acid and Terephthalic Acid in the pH Region around the Second pKa Values

Abstract In a reversed-phase high-performance liquid-chromatographic analysis phthalic acid and terephthalic acid were observed as two clearly separated peaks with a mobile phase of pHs around the second pKa values involving the monoanion and dianion species.

Preparative isolation of recombinant human insulin-like growth factor 1 by reversed-phase high-performance liquid chromatography

The isolation of recombinant human insulin-like growth factor 1 (rhIGF-1) is complicated by the presence of several rhIGF-1 variants which co-purify using conventional chromatographic media. These species consist primarily of a methionine-sulfoxide variant of the properly folded molecule and a misfolded form and its respective methionine-sulfoxide variant. An analytical reversed-phase high-performance liquid chromatography procedure using a 5-μm C 18, column, an acetonitrile-trifluoroacetic acid (TFA) isocratic elution, and elevated temperature gives baseline resolution of the four species. Using this analytical method as a development tool, a process-scale chromatography step was established. The 5-μm analytical packing material was replaced with a larger-size particle to reduce back-pressure and cost. Since the TFA counter-ion binds tightly to proteins and is difficult to subsequently dissociate, a combination of acetic acid and NaCl was substituted. Isocratic separations are not good process options due to problems with reproducibility and control. A shallow gradient elution using premixed mobile phase buffers at the same linear velocity was found to give an equivalent separation at low load levels and minimized solvent degassing. However, at higher loading there was a loss of resolution. A matrix of various buffers was evaluated for their effects on separation. Elevated pH resulted in a significant shift in both the elution order and relative retention times of the principal rh-IGF-1 variants, resulting in a substantial increase in effective capacity. An increase in the ionic strength further improved resolution. Several different media were evaluated with regard to particle size, shape and pore diameter using the improved mobile phase. The new conditions were scaled up 1305-fold and resulted in superimposable chromatograms, 96% recovery and ⪢99% purity. Thus, by optimizing the pH, ionic strength and temperature, a high-capacity preparative separation of rhIGF-1 from its related fermentation variants was obtained.

Leukotriene B4 generation and DNA fragmentation induced by leukocidin from Staphylococcus aureus: protective role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF for human neutrophils.

We studied the effect of leukocidin from Staphylococcus aureus V8 strains (Luk-PV) on the generation of Leukotriene B4 (LTB4) and its metabolites from human polymorphonuclear neutrophils (PMNs). Significant amounts of LTB4 were generated by PMNs after leukocidin exposure in a time- and dose-dependent manner, as shown by reversed-phase high-performance liquid chromatography analysis. In this regard, the S and F components of leukocidin acted synergistically. The calcium ionophore A23187 induced LTB4 generation, and the metabolism of exogenously added LTB4 into biologically less active omega-oxidated compounds was significantly decreased after leukocidin exposure. Priming of PMNs with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF prior to leukocidin exposure substantially increased toxin- and calcium ionophore A23187-induced LTB4 formation. The inhibitory effects of leukocidin on mediator release were accompanied by membrane damage and DNA fragmentation, which were both restored after pretreatment with GM-CSF. The data suggest that the presence of costimulatory priming factors such as GM-CSF or G-CSF in the microenvironment of an inflammatory focus determines the pathophysiological effects induced by S. aureus leukocidin.

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C 18 column. Those fractions eluting from the C 18 column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.

Reverse-Phase High-Performance Liquid Chromatography Analysis of Arachidonic Acid Metabolites in Plasma after Stimulation of Whole Blood ex Vivo

Following the stimulation of heparinized blood ex vivo, aliquots of plasma were denatured with organic solvents containing the internal standards prostaglandin (PG) B2 and 19-hydroxy-PGB2. Precipitated material was removed by centrifugation and the supernatants were directly analyzed by reverse-phase HPLC with on-line extraction and uv detection. Stimulation of blood with A23187 lead to the formation of both leukocyte and platelet arachidonic acid metabolites as the 5-lipoxygenase products leukotriene (LT) B4, 5-hydroxy-eicosatetraenoic acid (5-HETE), 20-hydroxy-LTB4 and 20-carboxy-LTB4, the 12-lipoxygenase product 12-HETE, and the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHTrE) were detected in plasma; in some plasma samples LTC4 and/or LTE4 were also detected. Stimulation of blood with zymosan lead to the synthesis of LTB4 and 5-HETE as major products and of 12-HETE. Recoveries of 20-hydroxy-LTB4, LTB4, 5-HETE, 12-HETE, and HHTrE added to plasma were high (≥90%) while those of LTC4 and LTE4 were lower (50-70%); however, washing of the precipitated protein pellet resulted in >90% recoveries for all metabolites including the cysteinyl-leukotrienes. Levels of arachidonic acid metabolites in native plasma samples stored at −20°C were stable for at least 28 days, while significant loss of material was observed over the same period of time in denatured plasma samples. Finally, we made the critical observation that the capacity for A23187- (but not zymosan-, ionomycin-, or LPS and FMLP-) induced arachidonic acid metabolite synthesis in blood decreased by 80% within 1 h of blood collection. The profiling of 5-lipoxygenase products in synovial fluid from an arthritic patient illustrates the applicability of the method to protein-rich matrices other than plasma. An application of the method is shown through the demonstration of the inhibition of LTB4 formation ex vivo following the in vivo administration of a LT synthesis inhibitor to rabbits.

Advances in Chromatography for Clinical Drug Analysis

Advances have been made in chromatography complement immunoassay for clinical drug analysis. Chromatographic theory shows that the minimum detectable mass is directly proportional to the square of column radius. Small internal diameter columns are capable of analyzing lower analyte concentrations, and high resolution is achievable in open capillary columns, concomitant with greatly reduced mobile phase consumption and waste. This review, based on the author's experience and literature, focuses on supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and selected high-performance liquid chromatography (HPLC) methodologies--microbore and direct-sample analysis (DSA) using commercially available Restricted Access Media (RAM) and REMEDi. In investigating the feasibility of SFC, the "normal-phase-like" selectivity of carbon dioxide was established, affecting the design of the extraction protocol and the elution order of drugs and metabolites. For example, the "more polar" tautomer eluted after FK-506, opposite to the order in the reversed-phase HPLC analysis. CE was investigated by Shihabi et al. for the analysis of pentobarbital and iohexol, while Evenson and Wiktorowicz performed preliminary evaluation of several therapeutic drug monitoring (TDM) drug groups. Innovation in HPLC column technology and hardware have greatly enhanced clinical drug analysis. Microbore column, offering enhanced mass sensitivity and high resolution, utilizes small sample size of 5 microliters of serum for the analysis of chloramphenicol. Commercially available RAM include internal surface reversed phase, shielded hydrophobic phase, and dual zone media, readily applicable for serum drug analysis without any sample preparation. Recently, an automated HPLC REMEDi offers urine and serum drug screening for toxicology.