High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent.

The assay of malondialdehyde (MDA) is widely used in clinical chemistry laboratories to investigate lipid peroxidation in oxidative pathologies. In the present work, the thiobarbituric acid (TBA) reaction was carried out on plasma, human erythrocytes and fibroblasts. The reagents used were those of the fluorimetry MDA kit manufactured by Sobioda. We have defined the application of this kit to high-performance liquid chromatography. This adaptation satisfied the criteria of good analytical practice. The detection limit was 2.5 pmol per injection. The retention time of the MDA-TBA2 peak (4.96 +/- 0.07 min) led to excellent resolution of the complex. The within-assay (6-12%) and between-assay (11-12%) precisions were satisfactory. The analytical recovery of MDA after spiking samples of human plasma with tetraethoxypropane standards varied from 70 to 100%. The mean lipoperoxide concentration determined in 32 healthy adults (20-40 years) was 1.04 +/- 0.23 mumol l-1 in plasma. Applied to the erythrocytes of fifteen laboratory workers, the method furnished physiological values of 0.59 +/- 0.21 mumol l-1. Concentrations were significantly higher in chronic renal dialysis patients (4.15 +/- 2.35 mumol l-1. The MDA content of fibroblasts cultured in standard medium was 0.38 +/- 0.04 mumol per g of protein and increased (5.78 +/- 1.38 mumol per g of protein) if the cells were grown in an iron-enriched medium. This accurate high-performance liquid chromatographic method for detection of MDA is the first one which can be applied to plasma, red blood cells and cultured cells. This technique will prevent false positives and should make inter-laboratory comparisons possible.     

Determination of dextromoramide in plasma and whole blood using high-performance liquid chromatography with ultraviolet absorbance detection.

Dextromoramide was determined in plasma and whole blood after solid-phase isolation by high-performance liquid chromatography using dextropropoxyphene as internal standard and ultraviolet detection at 215 nm. Owing to its good selectivity, sensitivity and reproducibility, the technique is available for forensic toxicology purposes as well as for clinical pharmacology. The concentrations of dextromoramide were determined in three cancer patients receiving intravenous treatment with one to three 5-mg daily doses. On the fourth day the plasma level was 13.85 +/- 3.27 ng ml-1 just before the first daily dose and 84.28 +/- 12.60 ng ml-1 30 min after dosing. The whole blood concentration, determined in one of the patients, was undetectable just before the dose and was 76 ng ml-1 30 min after dosing.     

Determination of metoprolol and two major metabolites in plasma and urine by column liquid chromatography and fluorometric detection

Metoprolol and its alpha-hydroxy metabolite were determined in plasma down to 2 nmol/l (S.D. 10-15%) after solvent extraction and bonded-phase liquid chromatography with fluorometric detection. The major metabolite with a carboxylic function was also measured in plasma when liquid-solid extraction on a column activated with dodecyl sulphate was applied. In urine the three components were assayed by direct injection of a diluted sample.     

On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-β fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.     

Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor-reporter gene yeast bioassay.

A recombinant cell yeast bioassay (RCBA) was applied to the generic measurement of bovine plasma oestrogen concentration. Samples were prepared by diethyl ether extraction of plasma following addition of [3H]17 beta-oestradiol as internal standard; organic and aqueous phases were separated by freezing (recovery 97.1 +/- 0.7%) and dried extract reconstituted in culture medium (recovery 31.4 +/- 4.5%). Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen receptor (hER) and a reporter construct comprising an hER response element regulating beta-galactosidase expression. The linearity of response for the analysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x - 0.111 (r2 = 0.9776, P < 0.0001). Inter-assay variation for endogenous oestrogen was 11.5% for > 1 pg ml-1. Plasma oestrogen concentrations for intact (n = 5) and castrated (n = 3) males were < 0.5 pg ml-1, and 3.7 +/- 2.6 pg ml-1 for luteal phase females (n = 10). Analysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17 beta-oestradiol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 +/- 2.1 pg ml-1 and 32.6 +/- 14.6 pg ml-1, respectively). Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gave concentrations (29.5 +/- 8.9 pg ml-1) not significantly different to those obtained by RIA. These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conjugated. These data indicate the potential of the RCBA to measure biologically active and physiological levels of plasma oestrogens in cattle. One potentially valuable application of this generic oestrogen assay could be in surveillance programmes to detect illegal use of anabolic oestrogens in live-stock where the identity of the analyte may be unknown.     

Membrane active plasma factor in multiple sclerosis: characterization and isolation by recycling isoelectric focusing

Recycling isoelectric focusing is a rapid, high resolution technique that has the capability of fractionating complex mixtures of proteins on a preparative scale on the basis of their isoelectric points (pIs). For this reason, it appeared to be an ideal tool to further characterize and isolate the surface active plasma component(s) which is abnormal in multiple sclerosis (MS). The normal control and the abnormal MS plasma components, or factors, proved to be stable under the conditions used in this technique, including deionization by electrodialysis, dialysis against distilled water, lyophilization and the presence of 3M urea and carrier distilled water, lyophilization and the presence of 3M urea and carrier ampholytes. The presence or absence of plasma factor activity was determined by incubating red blood cells in a test plasma, or plasma fraction, followed by the determination of the red blood cell electrophoretic mobility in the presence and absence of linoleic acid. Both the normal and MS factor had a pI of 4.0 +/- 0.1 under the conditions used. A high degree of purification was achieved and albumin was eliminated as a possible candidate for the factor(s).     

Purification of heparin cofactor II from human plasma

Heparin cofactor II (HCII) is an inhibitor of thrombin in human plasma whose activity is enhanced by heparin and dermatan sulphate. HCII was purified to homogeneity from normal human plasma with an overall yield of 7.5%. After treatment with barium chloride, precipitation with 50% saturated ammonium sulphate and dialysis of the resuspended precipitate against 0.02 M Tris-HCl (pH 7.4), the sample was chromatographed on a heparin-Sepharose CL 6B affinity column, DEAE-Sepharose CL 6B ion-exchange gel and an AcA 34 gel permeation column. For the final steps, a high-performance liquid chromatographic system was used which included ion-exchange chromatography on a Mono-Q column and gel permeation using a Superose column. The purified protein was homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific activity of purified HCII was 12.2 U/mg. The HCII activity was evaluated as antithrombin dermatan sulphate cofactor activity. A specific antiserum against HCII was raised in the rabbit.     

Determination of polyamines in hydrolysates of uremic plasma by high-performance cation-exchange column chromatography

The levels of putrescine, cadaverine, spermidine and spermine in uremic plasma were determined with an automatic polyamine analyzer with a 7.5 X 0.2 cm I.D. cation-exchange column using a stepwise sodium chloride gradient. All four polyamines were higher in ten patients with chronic renal failure than in eight normal subjects. The total polyamine content was also measured in the patients' plasma before and after maintenance dialysis; putrescine and spermidine levels were significantly lowered by the procedure.     

Sensitive high-performance liquid chromatographic assay using 9-fluorenylmethylchloroformate for monitoring controlled-release lidocaine in plasma

A sensitive high-performance liquid chromatographic (HPLC) assay using fluorescence detection for quantifying lidocaine levels in plasma (in the ng/ml range) was developed. This novel HPLC assay has made possible the simultaneous monitoring of lidocaine levels in coronary and peripheral plasma obtained after myocardial controlled-release matrix administration (0.92 mg/kg during 4 h) in the arrhythmic dog. The method employed extracts the drug from plasma using 1-chlorobutane and a subsequent derivatization with 9-fluorenylmethylchloroformate in acetonitrile at 110 degrees C. The derivative was chromatographed on a C18 reversed-phase column and measured with fluorescence detection (excitation 254 nm, emission 313 nm). N-Methylephedrine was found to be suitable as an internal standard, post-derivatization. The derivatization product of lidocaine was identified and characterized by mass spectral analysis. It was found to have a unique and reproducible dicarbamate structure, which was stable for at least three days at room temperature. The method was tested with human plasma as well as on dog plasma. Analytical recoveries were 88.6 +/- 3.6 and 77.4 +/- 3.0% (mean +/- S.E.), respectively, at levels ranging from 25 to 200 ng/ml. The lower detection limit was 1 ng/ml lidocaine. In conclusion, this rapid and convenient analysis was found to be suitable for the bioavailability pharmacokinetic assessment of lidocaine following low-dose regional drug administration.     

Validation and application of a high-performance liquid chromatography/tandem mass spectrometry assay for sumatriptan in human plasma

A sensitive and convenient high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) assay is described for the (5-HT(lB/lD)) receptor agonist sumatriptan in human plasma. Sumatriptan was recovered from plasma (81.8 +/- 6.8%) by liquid-liquid extraction. The mobile phase flow rate was 0.3 mL/min and consisted of methanol:water:formic acid (90:10:0.1, v/v/v). The analytical column (4.6 x 100 mm) was packed with Partisil C(8) (5 micro m). The standard curve was linear from 0.7 to 70.4 ng/mL (r(2) > 0.99). The lower limit of quantitation was 0.7 ng/mL. The assay was specific, accurate (percentage deviation from nominal concentrations were <15%), precise and reproducible (within- and between-day coefficients of variation <10.3%). Sumatriptan in plasma was stable over three freeze/thaw cycles and at room temperature for one day. The utility of the assay was demonstrated by following sumatriptan plasma concentrations in two healthy subjects for 8-12 h following a single 20 mg intranasal dose.     

Determination of catecholamines and their 3-O-methyl metabolites in mouse plasma

The determination of catecholamines and their 3-O-methyl metabolites in a single mouse plasma is necessary to understand the role of the sympathetic nervous activity, while the inactivation of catecholamines by catechol-O-methyltransferase indicates the activity of blood pressure regulation in animals. Here we report the basal catecholamines and their 3-O-methyl metabolite concentrations obtained from 15 microL of mouse plasma utilizing semi-microcolumn high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence detection system. The concentrations were 6.63 +/- 1.37 pmol/mL plasma, 0.49 +/- 0.10 pmol/mL plasma, 5.25 +/- 2.30 pmol/mL plasma, 3.23 +/- 0.84 pmol/mL plasma, 0.44 +/- 0.11 pmol/mL plasma, and 3.39 +/- 1.67 pmol/mL plasma for norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively (n = 5-7). Further, when blood pressure was reduced by minoxidil, plasma catecholamines were found to be significantly increased by the baroreflex-mediated response in mouse.     

The determination of sulphate in natural waters by inductively-coupled plasma emission spectrometry

Sulphate is determined simultaneously with other constituents by using inductively-coupled plasma emission spectrometry; the intensity of the 180.73-nm sulphur line is monitored. At a forward power of 1100 W, under compromise conditions, a 3 s detection limit of 0.08 mg l^-1 sulphate and a precision of 0.8% RSD at the 200 mg l^-1 sulphate level were obtained. A small spectral interference from calcium is overcome by software corrections, and good agreement is demonstrated between the proposed method and spectrophotometric sulphate measurements for a variety of natural waters including seawater.     

Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique

Arginine (ARG) is a substrate for endogenous nitric oxide (NO) production whereas its metabolite, asymmetric dimethylarginine (ADMA), acts as an inhibitor. Sufficient NO production is essential for cardiovascular key functions, thus elevated concentration levels of ADMA are related to a range of cardiovascular diseases. Owing to the lack of reliable methods for the measurement of ARG and ADMA in human plasma, concentration values determined with these methods can differ considerably. We present here a simple and very robust liquid chromatographic/mass spectrometric method for the determination of ARG and ADMA utilizing isotope-labeled internal standards. Sample preparation requires only protein precipitation; the analytes were derivatized with o-phthalaldehyde-mercaptoethanol and separated on a reversed-phase C(18) column with gradient elution. The analytes were detected with an electrospray ionization ion trap instrument working in the full-scan single mass spectrometry mode. Concentration values obtained with this method for healthy controls were ARG = 63.9 +/- 23.9 microM and ADMA = 0.355 +/- 0.066 microM, with a normal range for ADMA from 0.225 to 0.485 microM. The corresponding values for end-stage chronic renal failure patients are ARG = 48.1 +/- 18.5 microM, p < 0.01 and ADMA = 0.673 +/- 0.134 M, p < 0.001.     

Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography.

Assessment of lactate metabolism is of particular interest during exercise and in disease states such as diabetes, shock, and absorptive abnormalities of short-chain fatty acids by the colon. We describe an analytical method that introduces radio-active tracers and high-performance liquid chromatography (HPLC) to simultaneously analyze concentrations and specific activities (SAs) of plasma lactate. The HPLC conditions included separation on a reversed-phase column (octadecylsilane) and an isocratic buffer (30% acetonitrile in water). [3H]Acetate served as an internal standard. Lactate and acetate were extracted from plasma samples with diethyl ether following a pH adjustment to less than 1.0 and back-extracted into a hydrophilic phase with sodium carbonate (2 mM, pH greater than 10.0). Lactate is detected in the ultraviolet range (242 and 320 nm) by derivatization with alpha-bromoacetophenone. Control plasma samples were studied after an overnight fast for precision and analytical recovery. Calibration curves were linear in the range 0.18-6.0 mM (r = 0.92). The precision was 3% and the analytical recovery was 87%. The detection limit of the method was 36 pmol. Determination of lactate metabolism was performed in a patient with chronic congestive heart failure who was administered primed-continuous L-[U-14C]lactate (10 microCi bolus and 0.3 microCi/min continuously) during a 60-min rest period. Mean arterial lactate concentration and SA were 1.69 +/- 0.2 mM and 253.8 +/- 22 dpm/mumol, respectively. Systemic lactate turnover was 25.65 mumol/kg per min. Lactic acid systemic turnover, organ uptake and release rates can be accurately determined by isocratic HPLC.     

A new design of lead amalgam/lead sulphate electrode for potentiometric measurement of sulphate

A new lead amalgam/lead sulphate electrode has been developed which is easy to handle and can be used in a simple measuring cell. Its electrochemical characteristics have been tested in two different cell systems. The potentials obtained were very stable, and reproducible within +/-0.04 mV. The electrode exhibited a Nernstian response to sulphate and its calculated standard potential (-350.68 +/- 0.13 mV) agreed very closely with that recorded in the literature.     

Liquid chromatography-tandem mass spectrometry for the determination of a new oxazolidinone antibiotic DA-7867 in human plasma

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of a new ox-azolidinone antibiotic DA-7867, (S)-[N-3-(4-(2-(1-methyl-5-tetrazolyl)-pyridine-5-yl)-3- fl uorophenyl)-2-oxo-5-oxazolidinyl]methyl acetamide, in human plasma was developed. DA-7867 and internal standard, linezolid, were extracted from human plasma with ethyl acetate at acidic pH. A reverse-phase LC separation was performed on Luna C(8) column with the mixture of acetonitrile-ammonium formate (10 mm, pH 4.5; 35:65, v/v) as mobile phase. The analytes were determined using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The lower limits of quanti fi cation for DA-7867 was 2.5 ng/mL. The single liquid-liquid extraction quantitatively recovered DA-7867 and internal standard from plasma samples at the ranges of 82.2-86.7%. DA-7867 was stable in blank human plasma at room temperature for 24 h and following three freeze-thaw cycles.     

Determination of sodium tanshinone IIA sulfonate in plasma by liquid chromatography-electrospray ionisation-tandem mass spectrometry

Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIa, an important lipophilic component contained in Salvia miltiorrhizae. A simple, sensitive and robust quantification method for STS based on LC-ESI-MS/MS was developed and validated, and has been successfully applied to the pharmacokinetic study. Liquid-liquid extraction was used for extracting STS from biological samples, with a satisfactory recovery exceeding 75% at all test concentrations. Isocratic mobile phase consisted of 75% acetonitrile and 25% water containing 0.005% ammonia acetate (pH 3). Good retention and baseline separation for STS and the selected internal standard, diclofenac sodium, were obtained on a Shim-pack VP-ODS analytical column under this condition. The method was linear in the concentration range of 1-500 ng/mL. The intra- and inter-day precisions (RSD%) were within 9.0%. The deviation of the assay accuracies was within +/-10.0%. STS was proved to be stable during all sample storing, preparation and analytic procedures. With a lower limit of quantitation at 1 ng/mL, this method has been proved to be sensitive enough for the pharmacokinetic study of STS. The plasma profile of STS followed a single intravenous dosing was well fitted to a three compartmental model.     

Enzyme linked immunosorbent assay for beta-endorphin in human plasma.

We established a highly sensitive and specific double-antibody enzyme linked immunosorbent assay for beta-endorphin (beta-EP). For competitive reactions, the beta-EP-antibody was incubated with beta-EP standard (or sample) and beta-D-galactosidase-labeled beta-EP (delayed addition). Free and antibody-bound labeled antigen were separated by using an anti-rabbit immunoglobulin G coated immunoplate. The enzyme activity on the plate was fluorometrically determined. The minimal detection limit was approximately 0.4fmol/well (10 pmol/l). Using this assay system, beta-EP-like immunoreactivity (-LI) in human plasma was determined. The level of beta-EP-LI in extracted human plasma from 6 normal subjects was 2.44 +/- 0.68 pmol/l. High performance liquid chromatography analysis of the plasma of a normal subject revealed a single immunoreactive form which eluted with the same retention time as that of synthetic beta-EP.     

Simple, rapid and sensitive determination of plasma taurine by high-performance liquid chromatography using pre-column derivative formation with fluorescamine.

A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The plasma taurine contents of yellowtail fish, Seriola quinqueradiata, beef cattle, dairy cows and chickens were determined to be 125 +/- 54, 5.6 +/- 1.4, 2.2 +/- 0.7 and 20.0 +/- 9.6 micrograms/ml, respectively.     

Development of an HPLC method for the determination of salidroside in beagle dog plasma after administration of salidroside injection: application to a pharmacokinetics study

A simple RP-HPLC method was established for the determination of salidroside in dog plasma. Salidroside is one of the most active ingredients of Rhodiola L. The method had within-run precision values in the range of +/- 2.3 to +/- 9.1% (n = 5) and between-run precision in the range of +/- 3.2 to +/- 9.8%. A simple protein precipitation for salidroside extraction was processed using ACN at precipitant-to-plasma volume ratio (P-P ratio) of 3:2. The extraction recoveries of salidroside at seven concentrations were higher than 63.2%. There was a linear relationship between chromatographic area and concentration over the range of 0.83-520 microg/mL for salidroside in plasma (R = 0.9926). The LOQ (S/N = 10) of the method was 0.83 microg/mL. The method was applied in a study of the pharmacokinetics of salidroside injection in six beagle dogs. The major pharmacokinetic parameters of C(max), AUC(0-24), AUC(0-infinity), and t(1/2) of salidroside in beagle dogs after i.v. administration of a single 75 mg/kg (5 mL/kg) dose were 96.16 +/- 8.59 microg/mL, 180.3 +/- 30.6 microg h/mL, 189.3 +/- 32.1 microg h/mL, and 2.006 +/- 0.615 h, respectively.