High-performance liquid chromatography with amperometric detection applied to the screening of β-blockers in human urine

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of six β-blockers; atenolol, nadolol, timolol, metoprolol, oxprenolol, and alprenolol.The chromatographic separation was performed using a μBondapack C₁₈ column, a mobile phase of acetonitrile-water (40:60), containing 5 mM KH₂PO₄/K₂HPO₄ proved to be optimal at a 1.3 ml/min flow-rate, and a pH of 6.5. The temperature was optimized at 30±0.2°C. The amperometric detector, equipped with a glassy carbon electrode, was operated at 1300 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/ml), obtaining relative standard deviations lower than 5% at ppm levels and lower than 10% at ppb levels, and quantitation limits ranging from 15 ppb to 500 ppb.The method was applied to the screening of β-blockers in spiked urine samples, with a total elution time lower than 12 min, obtaining the best recoveries for timolol and metoprolol (never greater than 93%). These recoveries together with the low limits of quantitation achieved, allows its application to doping analysis in human urine.     

Quantification of retinyl-β-glucuronides in rat urine by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method is presented for the quantitation of the glucuronide conjugates of 4-oxo-all-trans-, 4-oxo-13-cis-, 13-cis-, 9-cis- and all-trans-retinoic acids in rat urine utilizing solid-phase extraction and gradient reversed-phase HPLC. The range of the R.S.D. (relative standard deviation) for both the inter- and intra-assay precision was 1.45,2–11.60%. The recovery of all retinoyl-β-glucuronides from rat urine ranged between 89 and 99%. The limit of detection was 0.01 μg/ml using 5 ml of rat urine. This method was applied to quantitate the amount of retinoyl-β-glucuronides produced in urine after the single and multiple oral administrations of 13-cis-, 9-cis- and all-trans-retinoic acids to rats.     

High-performance liquid chromatography and studies of neurophysin—neurohypophysial hormone pathways

High-performance liquid chromatography (HPLC) is being used extensively to characterize active polypeptides, precursor processing mechanisms, and cooperative peptide—protein noncovalent complexes in neuroendocrine pathways for neurohypophysial peptide hormones, oxytocin and vasopressin, and the hormone-associated proteins, neurophysins. Reversed-phase and ion-exchange HPLC polypeptide mapping have been used to detect the hormones, associated proteins, and other molecular forms containing these. This mapping but also ultimately to identify anatomical sites which contain the neurophysin/ hormone molecular pathways and to define the relatedness of polypeptide forms contained in different pathways. Reversed-phase HPLC also has provided a means to study proteolytic precursor processing, both to isolate synthetic and semisynthetic polypeptides and intermediates produced by these reactions. Finally, bioaffinity HPLC is being evaluated as a separatory and analytical tool. The latter includes its use to characterize the noncovalent peptide—protein and protein—protein interactions which occur among the molecular forms of the neurophysin/hormone pathways. These experiments typify the impact of HPLC for both analytical and preparative separations in studies of biologically active peptides and proteins.     

Assay of HA-966 in rat plasma by capillary gas—liquid chromatography with nitrogen-selective detection

A gas—liquid chromatographic method for the determination of the γ-aminobutyric acid-like drug 1-hydroxy-3-aminopyrrolidone-2 (HA-966) in plasma is described. HA-966 was converted into its diacetyl derivative Ac₂HA-966 with acetic anhydride. This compound could be suitably eluted from a capillary OV-17 support-coated open tubular column. A sensitive detection method was achieved by making use of nitrogen—phosphorus-selective flame ionization.     

The analysis of low levels of γ-glutamyltransferase activity by high-performance liquid chromatography with electrochemical detection

A method for the analysis of low levels of the enzyme γ-glutamyltransferase in biological samples by high-performance liquid chromatography with electrochemical detection is described. A γ-glutamyl moiety from glutathione is transferred by the enzyme to glycylglycine to produce a tripeptide which is assayed directly after a purification step using octadecylsilica. Confirmation of the method is by use of the inhibitor AT-125. The method is used to measure the level of enzyme activity in rodent tissues and in cultured cells.     

Assay of bromhexine in human plasma by capillary gas—liquid chromatography with nitrogen-selective detection and selected ion monitoring

A specific, sensitive method for the determination of bromhexine in human plasma is described. It comprises a selective extraction procedure and a specific determination with capillary gas—liquid chromatography and nitrogen-selective flame ionization detection. The detection limit of the assay is about 0.5 ng/ml. The specificity of the assay was checked by gas chromatography—mass spectrometry. The method is applied to the pharmacokinetics of bromhexine in humans.     

Micro-determination of plasma diphenylhydantoin by gas—liquid chromatography

A selective, sensitive and precise gas—liquid chromatographic method for the determination of diphenylhydantoin in micro samples of blood plasma is described. After a double extraction with chloroform containing an analogue of diphenylhydantoin as an internal standard, the drug and standard are N,N-dimethylated in alkaline aqueous solution with methyl iodide followed by extraction into acetone. The methylated derivatives are separated gas chromatographically and measured using a flame-ionization detector. The lowest concentration of diphenylhydantoin in plasma which can be measured in a 100-μl sample is 1 μg/ml, which is well below the normal therapeutic concentration of 10–20 μg/ml in plasma. The methylated derivatives of diphenylhydantoin and the internal standard have been identified by their proton magnetic resonance spectra and mass spectra.     

Practice of solid-phase extraction and protein precipitation in the 96-well format combined with high-performance liquid chromatography–ultraviolet detection for the analysis of drugs in plasma and brain

C₁₈ Empore 96-well extraction disc plates have been employed for the analysis of three drugs with different polarities in plasma in conjunction with HPLC–UV, rufinamide, ICL670 and an anticonvulsant agent (AA1) in an early stage of development. With the most polar compound (AA1), ion-pair extraction at pH 12 was applied. The method developed for the assay of AA1 in plasma was applied to its determination in brain using an Oasis HLB plate following homogenisation in a pH 7.4 buffer and protein precipitation with NaOH–ZnSO₄, thereby saving time for method development. Protein precipitation in the 96-well format with filtration of the precipitate was applied to the determination of ICL670, a highly protein-bound compound (>99.5%), with a good recovery (78%). Reversed-phase chromatography was applied using a short 5 cm column packed with 3 μm particles for the determination of ICL670 and AA1 and two parallel columns (15 cm long) for the determination of rufinamide. The methods were used routinely, one plate per analysis day being processed, resulting in increase in sample throughput and saving in solvents.     

High-performance liquid chromatographic determination of (−)-β-d-2,6-diaminopurine dioxolane and its metabolite,dioxolane guanosine,using ultraviolet and on-line radiochemical detection

(−)-β-d-2,6-Diaminopurine dioxolane (DAPD) and its metabolite dioxolane guanosine (DXG) have potent activity against hepatitis B virus and HIV, in vitro. A reversed-phase HPLC analytical method using UV and on-line radiochemical detection for the determination of DAPD and DXG in monkey serum and urine is described in this report. Retention times for DXG, DAPD and internal standard (2′,3′-didehydro-2′ deoxythymidine, D4T) were 5.0, 6.0 and 13.0 min, respectively. The extraction recovery was greater than 97% for DAPD and 94% for DXG. The limit of quantitation for UV detection was 100 ng/ml and 125 ng/ml for DXG and DAPD in monkey serum. The standard curves were linear from 0.1 μg/ml to 5 μg/ml for DXG and 0.125 μg/ml to 5 μg/ml for DAPD. For radiochemical detection, calibration curves of standard solutions of DAPD and DXG were linear in the range of 3500 Bq to 32 000 Bq and 7500 Bq to 60 000 Bq. The intra- and inter-day relative standard deviations were less than 7.2% using UV and less than 8.6% using on-line radiochemical detection. The HPLC method was applied to serum and urine samples collected from a male rhesus monkey that was administered 33.3 mg/kg DAPD with 200 μgCi of [³H]DAPD intravenously.     

Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography–tandem mass spectrometry detection

The tricarboxylic acid (TCA) cycle is an interface among glycolysis, lipid metabolism, and amino acid metabolism. Increasing interest in cancer metabolism has created a demand for rapid and sensitive methods for quantifying the TCA cycle intermediates and related organic acids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify the TCA cycle intermediates in a 96-well format after O-benzylhydroxylamine (O-BHA) derivatization under aqueous conditions. This method was validated for quantitation of all common TCA cycle intermediates with good sensitivity, including α-ketoglutarate, malate, fumarate, succinate, 2-hydroxyglutarate, citrate, oxaloacetate, pyruvate, isocitrate, and lactate using a 8-min run time in cancer cells and tissues. The method was used to detect and quantify changes in metabolite levels in cancer cells and tumor tissues treated with a pharmacological inhibitor of nicotinamide phosphoribosyl transferase (NAMPT). This method is rapid, sensitive, and reproducible, and it can be used to assess metabolic changes in cancer cells and tumor samples.     

Determination of terbutaline enantiomers in human urine by coupled achiral–chiral high-performance liquid chromatography with fluorescence detection

A coupled achiral–chiral high-performance liquid chromatographic system with fluorescence detection at excitation/emission wavelengths of 276/306 nm has been developed for the determination of the enantiomers of terbutaline, (S)-(+)-terbutaline and (R)-(−)-terbutaline in urine. Urine samples were prepared by solid-phase extraction with Sep-pak silica, followed by HPLC. The terbutaline was preseparated from the interfering components in urine on Phenomenex silica column and the terbutaline enantiomers and betaxolol were resolved and determined on a Sumichiral OA-4900 chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The precolumn was used to concentrate the terbutaline in the eluent from the achiral column before back flushing onto the chiral phase. For each enantiomer the assay was linear between 1 and 250 ng/ml (R²=0.9999) and the detection limit was 0.3 ng/ml. The intra-day variation was between 4.6 and 11.6% in relation to the measured concentration and the inter-day variation was 4.3–11.0%. It has been applied to the determination of (S)-(+)-terbutaline and (R)-(−)-terbutaline in urine from a healthy volunteer dosed with racemic terbutaline sulfate.     

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography—fluorescence detection method

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These guanidino compounds are separated on a 6 × 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.     

Metal ion-induced assembly of dipeptide-attached perylenediimide for fluorometric “turn on” detection of biologically important small molecule

A dipeptide-appended perylenediimide (PDI-CFF) fluorescent molecule was designed, synthesized, and characterized. Though the molecule does not dissolve in any individual solvent, it dissolves well in an organic/water mixed solvent system such as tetrahydrofuran/water. This new fluorescent molecule was self-assembled in a tetrahydrofuran/water mixture to form both nanofibrous network structures and a nano ring structure. It has shown nanofibril morphology by the interactions with ferric ions (PDI-CFF/Fe³⁺ system) with diminishing fluorescent property. Interestingly, L-ascorbic acid (LAA) interacts with the PDI-CFF/Fe³⁺ system, showing turn-on fluorescence. Another interesting feature is that the minimum detection limits for Fe³⁺ ions and LAA are at the submicromolar levels of 6.2 × 10⁻⁸ and 3 × 10⁻⁸ M, respectively. Moreover, the fluorescent (10 μM) signals can be monitored by the naked eye under handheld UV lamp irradiation at 365 nm, and this is very convenient for the real application. In this study, the molecule offers the opportunity for processing these sequential fluorescence responses in order to fabricate a implication logic gate that includes NOT, AND, and OR simple logic gates using chemical stimuli (ferric ions and LAA) as inputs and fluorescence emission at 536 nm as output. The detailed mechanism of interactions of Fe³⁺ with PDI-CFF and LAA with the PDI-CFF/Fe³⁺ system is vividly studied by using Fourier transform infrared (FT-IR) analysis and fluorescence. Moreover, this new molecule was reusable for several times without significant loss of its activity. The construction of logic gates using biologically important molecules/ions holds future promise for the design and development of new bio-logic gates.     

Liquid chromatography–fluorescence and liquid chromatography–mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody

Light exposure is one of several conditions used to study the degradation pathways of recombinant monoclonal antibodies. Tryptophan is of particular interest among the 20 amino acids because it is the most photosensitive. Tryptophan degradation forms several products, including an even stronger photosensitizer and several reactive oxygen species. The current study reports a specific peptide mapping procedure to monitor tryptophan degradation. Instead of monitoring peptides using UV 214 nm, fluorescence detection with an excitation wavelength of 295 nm and an emission wavelength of 350 nm was used to enable specific detection of tryptophan-containing peptides. Peaks that decreased in area over time are likely to contain susceptible tryptophan residues. This observation can allow further liquid chromatography–mass spectrometry (LC–MS) analysis to focus only on those peaks to confirm tryptophan degradation products. After confirmation of tryptophan degradation, susceptibility of tryptophan residues can be compared based on the peak area decrease.     

Determination of the α,β-adrenoceptor blocker YM-09538 in plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of the α,β-adrenoceptor blocker 5-{1-hydroxy-2-[2-(o-methoxyphenoxy)ethylamino]ethyl}-2-methylbenzenesulphonamide hydrochloride (YM-09538) in plasma, using 5-di-n-butylaminonaphthalene-1-sulphonyl chloride as a reagent for fluorescence labelling, is described. The detection limit is 20 ng/ml, which is sensitive enough to determine YM-09538 plasma levels after the oral administration of effective doses to dogs and humans.     

Determination of 17α-dihydroequilenin in rat,rabbit and monkey plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of total (unconjugated and conjugated) 71α-dihydroequilenin in male and female rat rabbit and male rhesus monkey plasma is described here. Plasma sample preparation involved hydrolysis with enzyme (Glusulase), addition of internal standard (14β-equilenin) and solvent extraction. The extracts were chromatographed on a C₆, 5-μm reversed-phase HPLC column and detection was accomplished with a fluorescence detector operated at an excitation wavelength of 210 nm and an emission wavelength of 370 nm. The assay was linear over a range of 2.5 to 100 ng/ml in male and female rat plasma, and 5 to 500 ng/ml in female rabbit and male and female monkey plasma. The method was specific, accurate and reproducible (percent differences <14.5; coefficients of variation <9.5%) in all matrices examined. The applicability of this method was successfully tested by quantifying total plasma concentrations of 17α-dihydroequilenin in ovariectomized female rats, ovariectomized female rabbits and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17α-dihydroequilenin sulfate intragastrically.     

High-performance liquid chromatographic resolution of (R,S)-α-alkyl-α-amino acids as diastereomeric derivatives

Summary A number (27) of racemic-alkyl--amino acids (AAA) were derivatized either witho-phthaldialdehyde (OPA) in combination withN-t-butoxycarbonyl-L-cysteine (Boc-Cys) orN-acetyl-L-cysteine (Ac-Cys), or withN ²-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The resolution of the diastereoisomers formed was investigated by reversed-phase (C₁₈) high-performance liquid chromatography (HPLC) using gradient elution conditions employing sodium phosphate buffers of pH 7.2 together with acetonitrile, and fluorescence detection at 344 nm (excitation) and 443 nm (emission) for the OPA/Boc-Cys or OPA/Ac-Cys derivatives. For the diastereomers formed by derivatization with Marfey's reagent triethylammonium phosphate buffers of pH 3.0 (pH 7.2 for acidic AAA) together with acetonitrile, and u.v. detection at 340 nm were used. Whereas with Marfey's reagent all diastereomers of AAA showed complete, or almost complete, resolution, only 8, or 11, respectively of the diastereomers formed by derivatization with OPA/Boc-Cys or OPA/Ac-Cys were resolved under the chromatographic conditions used.