A selective HPLC/RIA for the determination of budesonide

A combined HPLC/RIA procedure is described for the selective determination of budesonide (BUD) in plasma. The assay involves the extraction of plasma or serum samples with ethylacetate, consequent HPLC separation of intact budesonide from cross-reacting metabolites on a C₈ reversed phase column, collection of the budesonide containing fraction and determination of budesonide immunoreactivity with the budesonide antiserum. The method was accurate, sensitive (IC₅₀ value of 0.9 ng ml⁻¹) and reproducible (intra- and inter-day less than 15%) with a limit of quantification of 0.133 ng ml⁻¹ (RSD<25%). The evaluation of a limited number of clinical samples after oral administration of budesonide by both the HPLC/RIA procedure and a direct RIA using the same antiserum differed in average by a factor of 2, with the ratio of HPLC/RIA-RIA results declining as a function of time. Thus, this ratio might be a suitable indicator for probing for the ratio of budesonide and overall metabolites on a semi-quantitative level.     

Cyclosporin: Pharmacokinetics and detailed studies of plasma and erythrocyte binding during intravenous and oral administration

Summary On the basis that unbound concentration better correlates with response than total plasma or blood concentration, the inter- and intra-subject variability in the distribution of cyclosporin within blood and to plasma components was studied in renal transplant patients. Pharmacokinetic aspects were also studied.Blood samples were analysed from patients who received the drug both by a 72-h i.v. infusion and orally (7 mg·kg^–1 twice daily). Steady-state was reached within 18 h of starting the i.v. infusion; the plasma data were best fitted by a biexponential equation with half-times of 0.13–1.02 h and 4.3–13.9 h, associated with the two phases. The mean plasma clearance was 700 ml/min. Concentrations during the infusions measured by RIA and HPLC were comparable. Oral profiles showed rapid and extensive absorption. The peak plasma concentrations were 1460–1880 µg·l^–1 and occurred 2–4 h after dosing, with bioavailability estimates of 41–113%. Concentrations measured by RIA were higher than by HPLC.Blood-to-plasma concentration ratio measurements of cyclosporin at 37°C decreased with increasing plasma concentration and increased with haematocrit. Fraction unbound, measured by ultracentrifugation, was in the range 0.042–0.122 with an average of 0.068, and varied little in some patients but showed systematic changes with time in others. Cyclosporin binding was found to be related not only to the triglyceride but, more particularly, to the cholesterol-related lipoproteins in plasma. Monitoring cholesterol may be helpful in identifying patients with extremes in binding or with widely varying binding.     

Cytochrome P 450 2C9 phenotyping using low-dose tolbutamide

Objectives The hypoglycaemic drug tolbutamide is used for assessment of CYP2C9 activity in vivo. However, therapeutically active doses of 500 mg bear the risk of hypoglycaemia, and a tolbutamide-derived parameter based on a single plasma or urine concentration reflecting CYP2C9 activity accurately is lacking.Methods We examined tolbutamide and its metabolites 4-hydroxy-tolbutamide and carboxytolbutamide in plasma and urine of 26 healthy, male volunteers up to 24 h after intake of 125 mg tolbutamide using liquid chromatography–tandem mass spectrometry. CYP2C9 genotypes were determined by sequencing of exons 3 and 7. Raw plasma and urine data were compared with pharmacokinetic parameters, CYP2C9 genotypes, and data from a study in 23 volunteers with all six CYP2C9*1–*3 combinations who received 500 mg tolbutamide.Results Plasma clearance and tolbutamide plasma concentrations 24 h after drug intake reflected the genotypes: 0.85 l/h and 1.70 µg/ml (95% confidence interval, CI, 0.80–0.89 l/h and 1.50–1.90 µg/ml) for CYP2C9*1 homozygotes (n=15), 0.77 l/h and 2.14 µg/ml (95%CI, 0.67–0.88 l/h and 1.64–2.63 µg/ml) for *1/*2 genotypes (n=7), 0.60 l/h and 3.13 µg/ml (95%CI, 0.58–0.62 l/h and 2.68–3.58 µg/ml) for *1/*3 genotypes (n=3), and 0.57 l/h and 3.27 µg/ml in the single *2/*2 carrier. Natural logarithms of tolbutamide plasma concentrations 24 h after intake correlated to plasma clearance (r²=0.84, P<0.0000001). This correlation was confirmed in the comparison data set (r²=0.97, P<0.0000001).Conclusions A low dose of 125 mg tolbutamide can safely and accurately be used for CYP2C9 phenotyping. As a simple metric for CYP2C9 activity, we propose to determine tolbutamide in plasma 24 h after drug intake.     

Urinalysis for detection of chemically induced renal damage (3) — establishment and application of radioimmunoassay for lysozyme of rat urine

The radioimmunoassay (RIA) as a high sensitive detection method for rat lysozyme (LZM) was established and applied to determine LZM excretion in urine from rats treated with tubulotoxic chemicals in order to establish a sensitive method of detecting minor renal damage. Rat LZM which showed a single protein band on sodium dodecylsulfate polyacrylamide gel electrophoresis was purified by ion-exchange chromatography and gel filtration. The assay sensitivity of the established RIA using the purified rat LZM was 4–256 ng/ml rat LZM and was about 20 times the turbidity method. The concentration of LZM in normal rat urine was 76.2±6.0 ng/ml (mean ± SE, n = 50) using the RIA. In urine containing more than 100 ng/ml LZM, a high correlation between the values determined by the RIA and those by the turbidity method was observed. However, egg white LZM, human urinary LZM and guinea pig urinary LZM were not detectable by the RIA. Using the RIA, it was ascertained that urinary LZM excretion began to increase on day 5 in rats treated with gentamicin (15 or 30 mg/kg/day sc for 17 days), during the 6–9 h period in the rats treated with mercuric chloride (1 mg/kg sc), and during the 0–3 h period in the rats treated with p-aminophenol (1 mmol/kg sc). These significant increases in LZM excretion were not detectable by the turbidity method; therefore, it was concluded that this RIA for rat LZM was very useful for detection of minor renal damage.     

Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH): effect of aging on PAH excretion in the isolated perfused rat kidney

Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a commonly used marker of organic anion transport by the kidney. An analytical method for PAH using HPLC was developed. The method is simple, fast and requires a minimum amount of organic solvent. Sample preparation involved protein precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-phase phenyl column with UV detection at a wavelength of 254 nm. Mobile phase consisted of 0.1 M acetic acid and acetonitrile (99:1) at a flow rate of 1 ml/min. The assay was validated over a standard concentration range from 1 to 25 μg/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation <10%. The method was sensitive and showed linear response in peak height ratio (analyte:IS) over the concentration range studied (r²>0.99). The assay was used to study the effect of aging on PAH excretion in the isolated perfused rat kidney model. Experiments were conducted in kidneys from young (2–3 months, n=6), adult (6–9 months, n=5) and aged (12–16 months, n=3) male Sprague–Dawley rats at an initial drug concentration of 20 μg/ml. Significant differences in kidney function (e.g. glomerular filtration rate and glucose reabsorption) were observed in aged kidneys. Despite a 5-fold reduction in glomerular filtration rate, PAH renal clearance (kidney weight-corrected) decreased by only 2-fold in aged (2.2±0.42 ml/min per gram) compared to young (4.6±0.70 ml/min per gram, P<0.05) rats. Furthermore, renal excretion ratio was significantly higher in aged rats (27±8.0 vs. 15±5.0, P<0.05). These preliminary findings challenge the ‘Whole Nephron Hypothesis’ that assumes parallel reductions in renal filtration and secretory capacity secondary to disease or aging.     

Kinetics of ergotamine after intravenous and intramuscular administration to migraine sufferers

Summary The kinetics of ergotamine has been investigated in migrainous patients using a new, specific, sensitive HPLC assay (detection limit 100 pg/ml plasma). 10 patients were given ergotamine tartrate 0.5 mg i.v. and 5 of them received the same dose i.m. 2–3 weeks later. Blood samples were collected for up to 54 h following administration and the plasma concentration were analysed. After intravenous administration the plasma ergotamine declined rapidly, with an initial distribution half-life of 3 min followed by a mean terminal half-life of 1.86 h (range 90–155 min). The mean total plasma clearance was 11.0 ml kg^–1 min^–1, and the volume of distribution (V_d ) was 1847.6 ml kg^–1. Individual t_1/2 showed a positive linear correlation with the individual V_d . The intramuscular absorption of ergotamine was rapid and maximum plasma levels were usually obtained 10 min following administration. The biological availability was incomplete and variable at 46.6% (range 28.3–60.8%).     

The effect of liver cirrhosis on the pharmacokinetics of phenprocoumon

Summary Phenprocoumon was given orally to 9 patients with biopsy proven liver cirrhosis (dose range 0.12–0.25 mg/kg) and to 7 healthy volunteers (0.23 mg/kg). Concentrations of phenprocoumon were determined using HPLC in plasma and urine samples obtained for 6–7 days after drug administration. The binding of [³H]-phenprocoumon in plasma from all subjects was determined by equilibrium dialysis. Antipyrine plasma concentrations were determined spectrophotometrically following oral administration of antipyrine (1200 mg). The total body clearance of phenprocoumon was higher in the cirrhotic patients (1.64±0.16 ml/h/kg mean ± SEM) than in the healthy volunteers (0.90±0.07 ml/h/kg), however the free drug clearance was not significantly different in the patients (144±14 ml/h/kg) compared with normal (113±11 ml/h/kg). In contrast the clearance of antipyrine was much reduced in the cirrhotic group (17.5±2.9 ml/h/kg) compared with normal (35.6±3.9 ml/h/kg). The metabolic clearance of phenprocoumon via glucuronidation, is relatively unaffected during cirrhosis compared with antipyrine clearance via oxidation.     

Proof of the linearity of the pharmacokinetics of alinidine in man

Summary The pharmacokinetics of alinidine was investigated in two groups of volunteers: Group I (N=5) received on two different occasions single doses of¹⁴C-labelled drug given orally (40 mg) or intravenously (10 mg); Group II (N=6) received single oral doses 10, 30 or 90 mg dissolved in 20 ml water. The samples from Group I were analysed by two different and independent methods (RIA and counting total radioactivity). The results obtained by the two methods were identical, since the compound was not metabolized. The plasma concentrations and renal excretion data obtained from both groups were individually fitted to an open three compartment model. Independent of the route of administration and of the doses given, similar pharmacokinetic parameters were calculated for each group and each trial. The half lives of the distribution and elimination phases were t_1/2: 36–41 s, t_1/2 : 9.9–11.1 min and t_1/2: 2.7–3.8 h. There was a linear relationship between the dose administered and the resulting areas under the plasma concentration curves (AUC). Following a lag period (=0.19–0.22 h), the peak plasma concentration was reached 0.6–1.2 h after oral administration. Oral alinidine was 100% bioavailable.     

Determination of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in biological fluids by reversed-phase high pressure liquid chromatography

In this article a simple method for the determination of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in biological fluids is presented, based on reversed-phase ion-pair HPLC with UV detection at 254 nm. No complicated extraction procedure is needed. The stationary phase consists of a reversed-phase C18 stainless steel column and the mobile phase of 0.005M sodium acetate and 0.0025M pentanesulfonic acid sodium salt (PIC-B5) as an ion-pair reagent in water (pH 6.5). DHPG is a new antiviral drug in research of the treatment of cytomegalovirus infections. As a pilot study serum and urine samples of a few patients were investigated to gain an impression of the therapeutic range of DHPG. The method is suitable for routine assay of DHPG.     

The Bioavailability and Nonlinear Clearance of (–)-Carbovir in the Rat

The pharmacokinetics and bioavailability of (±)-carbovir, a carbocyclic nucleoside active against human immunodeficiency virus, have been described previously. To determine the bioavailability of (–)-carbovir, the biologically active enantiomer, four male Sprague–Dawley rats received 18 mg/kg of (–)-carbovir through the jugular vein and 54 mg/kg orally. Following the pilot studies, five rats were randomly assigned to receive (–)-carbovir in a three-way crossover design as either a single 18-mg/kg iv bolus, a single 54-mg/kg oral dose, or a single iv infusion of 18 mg/kg to achieve a target steady-state concentration (C _ss) of 1 µg/ml, the peak concentration after an oral dose. Blood and urine samples were analyzed by an improved ion-paired reversed-phase HPLC method with fluorescence detection. Blood concentrations of (–)-carbovir declined in a biphasic manner after the iv bolus dose. The terminal half-life was 116 and 106 min after the iv bolus and oral dose, respectively. The blood/plasma distribution ratio was approximately 1.0 in the range of 1 to 10 µg/ml of (–)-carbovir in blood. The free fraction in serum was concentration dependent. Significant differences in the renal, nonrenal, and total-body clearances after the iv bolus and iv infusion suggested nonlinear elimination of (–)-carbovir. The oral bioavailabilities derived from blood data were significantly different when the iv bolus was used as a reference rather than the iv infusion. However, the bioavailabilities were not significantly different when the total urinary excretion of unchanged (–)-carbovir after iv bolus or infusion was used as a reference. Concomitant saturation of renal and nonrenal clearances might explain these findings. The oral bioavailability was about 20% at concentrations approximating 1 µg/ml in blood.     

LC separation and induced fluorometric detection of rivastatin in blood plasma

An LC procedure suitable for quantitative analysis of pg ml⁻¹ concentrations of the HMG-CoA reductase inhibitor rivastatin in blood plasma was developed. The procedure involves an extraction step, chromatography on an ODS column, and fluorometric detection of a post-column photolytic decomposition product that was isolated and identified. The achieved quantitation limit (25 pg ml⁻¹) facilitated analysis of relatively low rivastatin concentrations in plasma that were observed after 100–300 μg oral doses of rivastatin. At 25 pg ml⁻¹ concentration the RSD ranged from 3.6 to 13.5% and mean deviation from the nominal value was 8.0%; at 8 ng ml⁻¹ the RSD range was 0.7–3.6% while the mean deviation was −1.8%. The concentrations obtained with the LC procedure were compared to the concentrations obtained with a specific but less sensitive capillary GC method and a radioimmunoassay (RIA) procedure. Concentrations obtained with the HPLC and GC procedures agreed within experimental error; the RIA concentrations were about 30% higher.     

Determination of the new aromatase inhibitor exemestane in biological fluids by automated high-performance liquid chromatography followed by radioimmunoassay

A procedure for the determination of exemestane, a new aromatase inhibitor, in biological fluids is described in this paper. Exemestane is extracted from human plasma and urine by solid-phase and liquid-liquid extraction, respectively. The test compound is then isolated from endogenous steroids, its metabolites and/or degradation products by HPLC. The exemestane-containing fraction is collected and its exemestane content measured by radioimmunoassay (RIA). The automated HPLC system allowed a high specificity and reproducibility of retention times, and eliminated almost all manual operations. The RIA allowed the accurate and precise measurement of 12 pg of exemestane/ml in plasma (inter- and intra-assay RSD=17.7 and 13.4%, respectively) and 25 pg/ml in urine (inter- and intra-assay RSD=14.5% and 8.7%, respectively). The recovery of the whole procedure was evaluated by comparison of the RIA calibration curve obtained in plasma or urine (after extraction and HPLC) with that obtained directly in RIA buffer (without extraction and HPLC). The calibration curves were practically superimposable, indicating that the recovery of the whole procedure was excellent. The method was validated in terms of reproducibility, recovery and precision in the range 10–500 pg of exemestane/ml of plasma and 20–1000 pg/ml of urine. Finally the plasma levels of exemestane in a postmenopausal healthy volunteer treated daily for 7 days with oral exemestane at a dose of 1 mg (the lowest dose administered in clinical trials) were monitored using the method here described. Exemestane was detectable in all plasma samples collected (up to 24 h after drug intake). Therefore the analytical method described here should be sufficiently sensitive and specific for the determination of exemestane in plasma and urine from clinical trials in which therapeutic doses of the drug (10–25 mg/day) are administered.     

Radioimmunoassay for mitoxantrone,a new antitumor agent

A sensitive and specific radioimmunoassay for mitoxantrone in serum has been developed. The procedure allows direct measurement of mitoxantrone in unextracted serum samples, by using antisera from rabbits immunized with mitoxantrone-BSA antigen. Tritiated mitoxantrone of high specific radioactivity (ca. 15 Ci/mmol) was used as a radio-tracer ligand. The assay allows the detection of as little as 50 pg/ml and the quantitation of 75 pg/ml in 0.5 ml serum samples. Standard curves were linear in the concentration range of 75–2500 pg/ml, at antiserum dilutions of 1:15,000. The assay shows good reproducibility: coefficients of variation of 3–6% were obtained by analyzing five samples/concentration at 75, 100, 250, 500, and 1000 pg/ml. There was no cross reactivity with the major metabolite in human serum, having concentrations of up to 10,000 pg/ml. Serum samples collected at various time intervals from rats dosed intravenously with mitoxantrone (0.5 mg/kg), were analyzed for unchanged mitoxantrone by RIA. The drug concentrations decreased from 32 ng/ml at 0.5 h to 0.45 ng/ml by 24 h after dosing.Mitoxantrone, a dihydroxyanthracenedione derivative (1), is an antitumor agent currently used in clinical trials with very encouraging results, especially in metastatic breast cancer, and low incidence of adverse reactions (2–4). The drug is being administered intravenously at doses up to 14 mg/m². Preliminary pharmacokinetic studies (to be published separately) indicate rapid distribution, followed by slow clearance rates from the tissues.The sensitivity of the currently available (HPLC) methods (5, 6) is of about 5–20 ng/ml in serum. The purpose of this work was to develop a more sensitive method, such as radioimmunoassay, which can be utilized in pharmacokinetic studies to measure mitoxantrone levels in biological fluids, over extended periods of time after dosing.

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A Pharmacokinetic Model of Intravenously Administered Hyaluronan in Sheep

Hyaluronan (HA; hyaluronic acid) is produced in the interstitium and reaches the blood circulation through the lymph. It is rapidly eliminated by means of specific receptors on liver endothelium. The elimination characteristics of intravenously administered HA were studied in 10 conscious sheep at the normal plasma HA concentration by injection of a ³H-labeled tracer and at a very high concentration by an i.v. infusion of unlabeled HA and simultaneous injection of a tracer dose of ³H-labeled HA. At a normal plasma HA concentration (0.12 ± 0.05 µg/ml; range, 0.072–0.228 µg/ml), the apparent T _1/2 of ³H-HA was 5.3 ± 1.1 min (range, 3.3–6.5 min). At higher plasma concentrations (range, 1.83–3.35 µg/ml), the apparent T _1/2 was considerably prolonged (range, 18.2–43.5 min). A one-compartment, nonlinear model was fitted to data obtained from the bolus-infusion study of unlabeled HA. The Michaelis–Menten constant, K _m , was 0.12 ± 0.04 µg/ml, indicating that a deviation from linear kinetics will occur when the normal plasma concentration is exceeded. The V _max was 0.062 ± 0.009 µg/ml/min. Three-dimensional surface plots showed that the plasma HA concentration and the total hepatic plasma flow influence the apparent metabolic clearance, extraction ratio, turnover, and T _1/2 of intravenously injected hyaluronan. There was a high correlation between T _1/2 as measured by the injected ³H-HA and T _1/2 calculated from the model (r = 0.96).     

Pharmacokinetics of vinorelbine in man

Summary The pharmacokinetics of vinorelbine has been investigated by a new HPLC method in 8 cancer patients receiving 8 weekly doses (30 mg·m^–2) administered by brief infusion (15 min).The plasma concentration-time curves showed a tri-exponential decay with a long terminal half-life (44.7 h) and a high volume of distribution (V_z=75.61·kg^–1). The concentrations after the 8th infusion were significantly lower than after the 1st infusion, but without significant modification of CL (1.28 l·h^–1·kg^–1) or AUC (0.80 mg·l^–1·h).The pharmacokinetic parameters exhibited wide inter-individual variations. The results are consistent with those of previous RIA studies, although the HPLC method appears to be more specific and more precise.     

Distribution,Metabolism and Tumoricidal Activity of Doxorubicin Administered in Sorbitan Monostearate (Span 60) Niosomes in the Mouse

Purpose. Encapsulation of doxorubicin in niosomes was sought as a route to tumour targeting and improved tumoricidal through the alteration of doxorubicin pharmacokinetics and metabolism. Methods. Doxorubicin niosomes (10 mg kg^–l doxorubicin) prepared from sorbitan monostearate (Span 60), cholesterol and choleth-24 (a 24 oxyethylene cholesteryl ether) in the molar ratio 45:45:10 were administered intravenously to female NMRI mice bearing the MAC 15A subcutaneously implanted tumour. Plasma doxorubicin was fractionated by gel filtration and quantified by HPLC with fluorometric detection as niosome-associated doxorubicin and released doxorubicin. Tumoricidal activity of the formulation was assessed by the intravenous injection of 5 mg kg^–1 and 10 mg kg^–1 doxorubicin niosomes to male NMRI mice bearing a 6 day old MAC 15A tumour. Results. At least 90% of the plasma doxorubicin was associated with the niosome fraction 4 h after dosing, and 50% was still associated after 24 h. The clearance of doxorubicin released from the niosomes was about 10 fold greater than the clearance of niosomal doxorubicin (176.5 mL h^–l and 16.2 mL h^–1, respectively). The area under the plasma level-time curve increased 6 fold when doxorubicin was administered in niosomes, compared to doxorubicin solution (66.0 µg.h mL^–l and 10.3 µg.h mL^–1, respectively). The area under the tumour level time curve was increased by over 50% by the administration of doxorubicin in niosomes when compared to the drug administered in solution (58.6 µg.h mL^–l and 34.3 µg.h mL^–1, respectively). There was no statistically significant difference between levels of the drug in the heart when niosomal doxorubicin or doxorubicin solution were administered. Doxorubicin metabolites, namely doxorubicinol and the aglycones doxorubicinone, doxorubicinolone and 7-deoxydoxorubicinone, were found associated with the niosomes in the plasma, possibly due to their adsorption to the vesicle surface once formed outside the niosome. Overall metabolite levels in the liver were increased when doxorubicin niosomes were administered compared to the drug in solution. A 5 mg kg^–1 injection of doxorubicin niosomes produced a terminal mean tumour weight that was similar to that obtained from animals administered 10 mg kg^–1 doxorubicin solution. Conclusions. Modest tumour targeting was achieved by the delivery of doxorubicin in sorbitan monostearate niosomes, increasing the tumour to heart AUC^0–24 ratio from 0.27 to 0.36 and a doubling of tumoricidal activity. The overall level of doxorubicin metabolites was also increased.     

Stereoselective hemodynamic effects of (R)-and (S)-propranolol in man

Summary In a randomized, double-blind, placebo-controlled, cross-over study 24 healthy volunteers were examined before and 2 h after oral administration of 80 mg (R,S)-, 40 mg (R)- and 40 mg (S)-propranolol · HCI; 8 of them received placebo in an additional run. During exercise on a bicycle ergometer and a rest period the rate pressure product was decreased by 80 mg (R,S)-propranolol · HCl (–32.8%p < 0.0001) and 40 mg (S)-propranolol · HCl (–32.3%;p < 0.0001), whera 40 mg (R)-propranolol · HCl as well as placebo showed no effect. corresponding binding inhibition experiments using (–)-(¹²⁵I)iodocyanopindolol in a sarcolemma-enriched cardiac membrane preparation yielded a eudismic ratio of 179 for (S)- over (R)-propranolol. 2 h after oral application, stereospecific HPLC analysis revealed different individual concentrations in plasma of (R)-(22.3 ± 21.7 ng/ml) and (S)-propranolol (30.4 ± 26.9 ng/ml) when 80 mg of (R,S)-propranolol · Hcl was administered. The plasma levels were similar when 40 mg of the pure enantiomer of (R)- (22.7 ± 20.3 ng/ml) or (S)-propranolol · HCl (28.7 ± 22.5 ng/ml) was applied. (R)- and (S)-propranolol are two substances with different pharmacodynamic and pharmacokinetic properties. As there are methods available to produce the optically pure enantiomers, they should be used rather than the racemic mixture.     

Liquid Chromatographic Analysis of Di(2-ethylhexyl) Phthalate: Application to Pharmacokinetic Studies in the Mongrel Dog

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of di(2-ethylhexyl) phthalate (DEHP) in serum or plasma. Plasma DEHP concentrations that were measured by HPLC in specimens obtained from hemodialysis patients were in good agreement with corresponding concentrations that were measured by gas chromatography with selected ion monitoring (GC-SIM) (r ² = 0.996). Plasma DEHP concentrations were measured after intravenous DEHP administration (1.2–4.4 mg DEHP/kg body weight) to determine the effect of bilateral ureteral ligation on DEHP elimination in the mongrel dog. DEHP plasma clearance (6.3 ml/min/kg), steady-state distribution volume (0.2l/kg), and terminal half-life (50 min) were unchanged in two dogs following bilateral ureteral ligation. DEHP terminal half-life and steady-state distribution volume were substantially smaller (25- to 70-fold) than reported previously in the rat or dog.     

Kinetic analysis of covalent binding between N-acetyl-L-cysteine and albumin through the formation of mixed disulfides in human and rat serum in vitro

Purpose. Covalent binding between N-acetyl-L-cysteine (NAC) and albumin was evaluated kinetically by conducting in vitro experiments. Methods. After ¹⁴C-NAC was incubated with human or rat serum, the solution was analyzed by anion-exchange HPLC. The albumin-bound ¹⁴C-NAC was quantified by measuring the radioactivity in the albumin fraction. Results. Ultraviolet chromatograms and/or radiochromatograms indicated the presence of a stable covalent bond between ¹⁴C-NAC and either human or rat albumin. By analyzing the time dependence of this protein binding in serum, the first-order binding and dissociation rate constants (k_on and k_off) were obtained. The serum was treated in a CO₂ incubator to avoid oxidative interference, and the initial rates were determined separately. The k_on values obtained were 0.33 (h^–1) and 0.48 (h^–1) for human and rat serum, respectively. L-Cysteine was required to initiate the dissociation of ¹⁴C-NAC bound to albumin. Following the addition of appropriate amounts of L-cysteine, the k_off values were determined to be 0.30-1.0 h^–1 and 0.54-1.4 h^–1 for human and rat serum, respectively. Conclusions. The k_on and k_off values obtained for rat serum were in good agreement with the in vivo plasma protein binding kinetics of NAC in rats, indicating the reliability of this in vitro method for evaluating protein binding. No species differences in protein binding kinetics were found between human and rat serum.     

Ultra-performance liquid chromatography-tandem mass spectrometry for the determination of lacidipine in human plasma and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–ESI-MS/MS) method was developed and validated for the quantification of lacidipine in human plasma. With nifedipine as an internal standard, sample pretreatment involved a simple liquid–liquid extraction with tert-butyl methyl ether of 1 ml plasma. The analysis was carried out on an Acquity™ UPLC BEH C₁₈ column (50 mm × 2.1 mm, 1.7 μm) with flow rate of 0.28 ml/min. The mobile phase was 30 mM ammonium acetate buffer–acetonitrile (18:82, v/v, pH 5.5). The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 0.025–10.000 ng/ml, with a lower limit of quantification of 0.025 ng/ml. The intra- and inter-day precision (R.S.D.) values were below 15% and accuracy (RE) was −12.7% to 11.9% at all QC levels. The method was successfully applied to a clinical pharmacokinetic study of lacidipine in healthy volunteers following oral administration.