Quantitative liquid chromatographic determination of sanguinarine in cell culture medium and in rat urine and plasma

Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10-2000 ng ml(-1). The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml(-1) in cell culture medium and in rat urine and 100 ng ml(-1) in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.     

Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP)

Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.     

A rapid,sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 mm sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10³ times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.     

Hydroxyapatite high-performance liquid chromatography: column performance for proteins

Hydroxyapatite columns for high-performance liquid chromatography that are reusable for a long time were developed; performance tests were carried out by using several types of protein. Using a high flow rate, a sharp chromatographic peak can be obtained for a homogeneous molecule. A very high level of chromatographic separation can be achieved by decreasing the slope of the gradient and increasing the total column length at the same time.     

Determination of urinary 4-hydroxy-3-methoxyphenylethylene glycol in man by high performance liquid chromatography with electrochemical detection

A reverse phase high performance liquid chromatographic method for determining levels of 4-hydroxy-3-methoxyphenylethylene glycol (MHPG) in human urine that is virtually free of all interfering peaks has been developed. After addition of a homologous internal standard, enzymatic hydrolysis is performed. Samples are then placed onto columns containing AG1-X8, and the MHPG is collected in a phosphate buffer wash of the column. After ethyl acetate extraction and evaporation of the organic solvent, the dry residue is redissolved in mobile phase, and injected onto a reverse phase column. Results obtained with this assay were almost identical (101±5.6%, mean±SD, n=6) with those obtained using a gas chromatography - mass spectrometry (GCMS) assay.     

Identification of delta-guanidinovaleric acid in human urine

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

Quantification of plasma phenylethylamine by combined gas chromatography/electron capture negative ion mass spectrometry of the N-acetyl-N-pentafluorobenzoyl derivative

An ultrasensitive method capable of detection and quantification of beta-phenylethylamine in 1 ml of human plasma has been developed using gas chromatography/electron capture negative ion mass spectrometry. Phenylethylamine and tetra-deutero phenylethylamine internal standard in plasma were acetylated, extracted into organic solvent and then further acylated with pentafluorobenzoyl chloride. The N-acetyl-N-pentafluorobenzoyl-phenylethylamines were detected by high-resolution single ion monitoring of the molecular ions. Normal plasma levels were found to be 41.5 +/- 10.7 pg ml-1, in accordance with results of a previous high-performance liquid chromatographic method.     

A study of the parameters affecting flow gradient analysis of catecholamines, DOPA and DOPAC by ion pair liquid chromatography with electrochemical detection

A general method for the determination of norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylalanine and 3,4-dihydroxyphenylacetic acid is described. It employs the generation of a flow gradient during the analysis by ion-pair reversed phase liquid chromatography. The method allows a reduction of the analysis time maintaining high resolution of the peaks close to the solvent front. The effect of the flow gradient on column efficiency, detector response and retention are evaluated. The application to specimens of amniotic fluid, urine, human plasma and rat brain tissue is shown.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Effects of Hexane in Supercritical Fluid Chromatography for the Separation of Enantiomers

Supercritical fluid chromatography (SFC), operated in conventional mode, is normally recognized as normal phase chromatography, and uses a solvent combination of supercritical CO₂ and alcohols to separate compounds. Hexane, a commonly used solvent in normal phase liquid chromatography (NP‐LC), is rarely used in SFC and, in some cases, is added to the organic modifiers to increase liquid content in order to achieve better efficiency in preparative SFC for poorly retained compounds. Although hexane is believed to have similar solvent strength to that of supercritical CO₂, its effects on the enantioseparation in SFC is largely unknown. To understand the chromatographic effects of an apolar solvent, such as hexane in SFC, we compared the chromatographic behaviors of 35 chiral compounds using a parallel SFC method under traditional SFC mode of only “pure” alcohol‐CO₂ to that of hexane‐assisted SFC (HA‐SFC), which uses mixtures of alcohol and hexane (as cosolvents) and CO₂. We observed that, in some cases, hexane behaves just like supercritical CO₂, where replacement of a portion of CO₂ with hexane does not significantly change retention times or resolution of the peaks. In many cases, however, addition of hexane in mobile phases does affect chromatographic behavior of one or both enantiomers. Such effects might provide opportunities for separation of some enantiomers. Chirality 28:192–198, 2016. © 2016 Wiley Periodicals, Inc.     

Liquid chromatography-tandem mass spectrometry analysis of oleuropein and its metabolite hydroxytyrosol in rat plasma and urine after oral administration

We describe a liquid chromatography-electrospray ionisation tandem mass spectrometry method for the qualitative and quantitative determination of the secoiridoid oleuropein and its bioactive metabolite hydroxytyrosol in rat plasma and urine. Samples were prepared by liquid-liquid extraction using ethyl acetate with a recovery for both compounds of about 100% in plasma and about 60% in urine. The chromatographic separation was performed with a RP-ODS column using a water-acetonitrile linear gradient. The calibration curve was linear for both biophenols over the range 2.5-1000 ng/ml (LOD 1.25 ng/ml) for plasma and 5-1000 ng/ml (LOD 2.5 ng/ml) for urine. Plasma concentrations of oleuropein and hydroxytyrosol were measured after oral administration of a single dose (100 mg/kg) of oleuropein. Analysis of treated rat plasma showed the presence of unmodified oleuropein, reaching a peak value of 200 ng/ml within 2 h, with a small amount of hydroxytyrosol, whereas in urine, both compounds were mainly found as glucuronides.     

Vitamin D in plasma: quantitation by a nonequilibrium ligand binding assay

The concentration of vitamin D was determined in human and bovine plasma samples under various physiological and nonphysiological conditions using a nonequilibrium ligand binding assay. Prior to ligand binding analysis the vitamin D in the plasma organic extracts was purified using chromatographic procedures involving Lipidex-5000 and high performance liquid chromatography. The use of a nonequilibrium assay system greatly increased the sensitivity of our assay allowing for a minimum volume of the initial plasma sample. The vitamin D levels in plasma responded to increased sun exposure as well as to the intoxication with vitamin D3. Analysis of a plasma sample from a vitamin D-deficient patient revealed that lipid interference was not a factor in this ligand binding assay.     

Validation of high-performance liquid chromatography-tandem mass spectrometry assays for the quantification of eribulin (E7389) in various biological matrices

This paper presents specific and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assays for the quantification of the novel anticancer agent eribulin in human plasma, whole blood, urine and faeces. These assays, developed to support clinical pharmacological studies with the drug, quantify eribulin concentration ranges of 0.2-100ng/mL for plasma, 0.5-100 ng/mL for whole blood and urine and 0.1-25 μg/g for faeces, using sample volumes of 500 μL or 250 μg (faeces). Samples were prepared with liquid-liquid extraction, separated on a C18 column with gradient elution and analysed with a triple quadrupole MS, in positive ion mode. A structural analogue of eribulin was used as internal standard for the quantification. The assays were linear with correlation coefficients (r(2)) of 0.99 and better, whereby the deviation from nominal concentrations ranged from -8.2 to 8.9% with CV values of maximally 14.2%. Stability assessments demonstrated that eribulin is stable at -20°C in plasma, whole blood, urine and faeces for at least 38, 4, 10.5 and 5 months, respectively. In conclusion, the validation results show that the assays are specific and accurate and can therefore adequately be applied to support clinical studies of eribulin.     

High-performance liquid chromatographic analysis of dipyridamole in plasma and whole blood

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of dipyridamole in plasma and whole blood. The method involves a single extraction of an alkalinized sample with diethyl ether followed by evaporation of the organic solvent and ion-pair chromatography using fluorescence detection. The lower limit of sensitivity for dipyridamole is 1 ng/ml. Concentrations of dipyridamole between 1 and 500 ng per sample are measured with an average coefficient of variation of 4.5% in plasma and 7.4% in whole blood.     

Quantitative determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry employing an automated liquid-liquid extraction based on 96-well format plates. Application to a bioequivalence study

An automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for quantitative determination of donepezil in human plasma. 150 MicroL of plasma samples were placed in 2.2 mL 96-deepwell plates and both donepezil and loratadine (IS) were extracted from human plasma by liquid-liquid extraction (LLE), using hexane as the organic solvent. Robotic liquid handling workstations were employed for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. The method developed, includes a sample analysis performed by reversed phase LC-MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The chromatographic run time was set for 2.0 min with a flow rate of 0.7 mL/min in a C18 analytical column. The method was significantly sensitive, specific, accurate and precise for the determination of donepezil in human plasma and had the shortest run time. The curve was proven to be linear for the concentration range of 0.1-100 ng/mL. After validation, the method was applied to the rapid and reliable quantitative determination of donepezil in a bioequivalence study after per os administration of a 5mg donepezil tablet.     

The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C(18)-columns (Synergi 2.5 μm, Hydro-RP, 100 ?, the length of 100mm) with an inner diameter of 2 or 3mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.     

Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry

A rapid, sensitive method using liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C₁₈ reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25–50 μg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation.     

Rapid and simple measurement of serotonin N-acetyltransferase activity by liquid biphasic diffusion assay

We report here a rapid, simple, and accurate method to assay for serotonin N-acetyltransferase (NAT) activity. This assay relies on the selective diffusion of radiolabeled acetyltryptamine into a water-immiscible scintillation fluid. Unlike organic solvent extraction, thin-layer chromatography, or high performance liquid chromatography, the separation of acetyltryptamine from acetyl CoA and tryptamine is not required in the method. Moreover, the limit of sensitivity is less than 4 pmol of N-acetyltryptamine formed per sample. Enhancement of NAT activity upon β-adrenergic receptor stimulation in the rat pineal gland was clearly detected with this method. In addition, the NAT activity measurements obtained with this method agreed quantitatively in the pineal gland and other brain tissues with the conventional organic solvent extraction method. The results suggest that this liquid biphasic diffusion assay is applicable to the detection of NAT activity in tissues and cells.     

Bioanalysis of drugs by liquid-phase microextraction coupled to separation techniques

The demand for automation of liquid-liquid extraction (LLE) in drug analysis combined with the demand for reduced sample preparation time has led to the recent development of liquid-phase microextraction (LPME) based on disposable hollow fibres. In LPME, target drugs are extracted from aqueous biological samples, through a thin layer of organic solvent immobilised within the pores of the wall of a porous hollow fibre, and into an microl volume of acceptor solution inside the lumen of the hollow fibre. After extraction, the acceptor solution is subjected directly to a final analysis either by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), mass spectrometry (MS), or capillary gas chromatography (GC) without any further treatments. Hollow fibre-based LPME may provide high enrichment of drugs and excellent sample clean-up, and probably has a broad application potential within the area of drug analysis. This review focuses on the principle of LPME, and recent applications of three-phase, two-phase, and carrier mediated LPME of drugs from plasma, whole blood, urine, and breast milk.