Determination of plasma tocopherols by high-performance liquid chromatography with coulometric detection

An HPLC procedure has been developed for tocopherol determination with coulometric detection in human serum samples. Eluent optimization and foreign peak identification (bilirubin) by mass spectrometry are described. An extraction procedure gave yields around 98% with 1.3% coefficient of variation, and the calibration ranged from 0.1 to 200 mg/l with a correlation coefficient of 0.999. The detection limit achieved for vitamin E was 60 pg (3 ng/ml).     

Determination of naloxone in human plasma by high-performance liquid chromatography with coulometric detection

Naloxone, the analyte and the internal standard, sumatriptan, are extracted from plasma using solid-phase extraction columns. Chromatography and detection are performed using isocratic reversed-phase high-performance liquid chromatography (HPLC) with coulometric end-point detection. The standard curve was linear over the range 0–50 ng/ml of naloxone in plasma. The reproducibility, the coefficient of variation (C.V.) of the method over the range of the standard curve was 6.2–11.2%. The recovery averaged 90.4±8.9%. A plasma profile following i.v. administration of naloxone in one normal healthy volunteer is presented.     

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid—liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 μg/l (500-μl sample). Reproducible recoveries in urine were obtained if 10–50 μl of sample were processed supplemented with 500 μl of blank plasma.     

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0–100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9–7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.     

Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.     

Determination of caffeic acid in rabbit plasma by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method involving UV detection was developed for determination of caffeic acid in rabbit plasma. A Lichrosphere CN column (250 mm × 4 mm I.D., 5 μm) was used as the stationary phase and the mobile phase consisted of 2% acetic acid solution at a flow-rate of 1.0 ml/min. The UV absorbance was monitored at 320 nm. The plasma sample was acidified by the addition of 0.01 parts of concentrated phosphoric acid (85%) to maintain caffeic acid stability. After a simple clean-up procedure, the limit of quantitation achieved was 0.1 μg/ml, and the standard curve was found to be linear over the concentration ranges of 0.1–2.0 μg/ml and 0.1–40 μg/ml. The coefficient of variation for within- and between-run precision and accuracy was less than 10%, and the recovery was 82.3%.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

Determination of iodide in serum and urine by ion-pair reversed-phase high-performance liquid chromatography with coulometric detection

An HPLC method is described for the determination of iodide in serum and urine using ion-pair chromatography with coulometric detection. After adding hexadecyltrimethylammonium chloride, the ions pairs formed with the iodide in the sample are extracted using an organic solvent. The solvent is then evaporated and the dry residue obtained is mixed with an appropriate volume of mobile phase so as to concentrate the sample prior to injection into the chromatograph. For a sample of 0.5 ml of serum, the method features a limit of detection (signal-to-noise ratio of 3) of 0.2 μgl⁻¹, sufficient to be applied in paediatric assays for the diagnosis of both iodide deficiency and excess.     

Determination of maprotiline in plasma by high-performance liquid chromatography with chemiluminescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of maprotiline, an antidepressant, in plasma. After a single-step extraction from plasma (100 μl) with n-hexaneisoamylalcohol (19:1, v/v), the drug and desipramine (internal standard) are converted into their chemiluminescent derivatives by reaction with 6-isothiocyanatobenzo[g]phthalazine-1,4(2H,3H)-dione, a new chemiluminescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSKgel ODS-80, using isocratic elution with acetonitrile-100 mM acetate buffer (pH 3.2), and produced chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline medium. The detection limit for maprotiline added to plasma is 0.36 pmol (0.1 ng)/ml plasma (1.5 fmol on column), at a signal-to-noise ratio of 3.     

Determination of formaldehyde in blood plasma by high-performance liquid chromatography with fluorescence detection

An HPLC method was developed for the determination of formaldehyde in human blood plasma. The method was based on the determination of the fluorescent product of the chemical reaction between formaldehyde and ampicillin. A 0.2-ml aliquot of blood plasma was reacted directly with ampicillin under acidic and heating conditions. The reaction product was extracted from the matrix with diethyl ether and analyzed by reversed-phase HPLC with fluorescence detection. Recoveries of spiked formaldehyde at the low ppm (μg/ml) level were between 93% and 102% with relative standard deviations less than 8%. The limits of detection and quantitation of formaldehyde in blood plasma samples were 0.46 μg/ml and 0.87 μg/ml, respectively.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40°C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.     

Determination of dihydroergotamine in human plasma by high-performance liquid chromatography with fluorescence detection

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1–10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

Determination of amiprilose in human plasma by high-performance liquid chromatography with fluorimetric detection

A reversed-phase HPLC method to quantify amiprilose in human plasma is described. The method involves liquid–liquid extraction of amiprilose and the internal standard from plasma. The extracted compounds are derivatized with 1,8-naphthalic dicarboxylic acid using 2-chloro-1-methylpyridinium iodide as a coupling reagent. The derivatized products are separated on a reversed-phase column and monitored fluorimetrically using 280 nm and 340 nm as excitation and emission wavelengths, respectively. The derivatized products which exhibit two peaks on chromatogram, are shown to be the interconvertible isomers. This assay has been used in pharmacokinetic studies of amiprilose in humans.     

Determination of succinylcholine in human plasma by high-performance liquid chromatography with electrochemical detection

An alternative HPLC method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine in human plasma is described. Drug spiked plasma and patient plasma samples were extracted using a C₁ solid-phase cartridge. Succinylcholine was separated on a Cyano column and quantitated using electrochemical detection at a potential of 450 mV and 750 mV. Mobile phase consisted of a mixture of phosphoric acid–acetonitrile–methanol (45:35:25) adjusted to an apparent pH of 5. Standard curves for the quantitation were linear in the range of 250–8000 ng/ml. Between-day and within-day relative standard deviations were 5.1% and 1.7%, respectively. Mean drug recovery and accuracy was 68% and 104%, respectively.