Measurement of plasma concentrations of isosorbide dinitrate

Concentrations of the vasodilator isosorbide dinitrate (ISDN) in human plasma can be measured with good sensitivity (about 0·2–0·5 ng ml^?1) using electron-capture gas chromatography after a one-stage extraction. The mean recovery of ISDN from plasma was 83 per cent ± 9 standard deviation (S.D.). The precision of the method for the measurement of ISDN in plasma ranged from ± 14 per cent at 1 ng ml^?1 to ± 7 per cent at 5 ng ml^?1 to ± 4 per cent at 50 ng ml^?1. The 95 per cent confidence limits of the least-squares regression calibration line forced through the origin were ± 100 per cent at 1 ng ml^?1, ± 11 per cent at 10 ng ml^?1, and ± 8 per cent at 30 ng ml^?1. The method has been used to assay many samples withdrawn after doses of drug at therapeutic levels to normal subjects.     

LC-MS-MS analysis of 2-pyridylacetic acid,a major metabolite of betahistine: application to a pharmacokinetic study in healthy volunteers

1.?A sensitive liquid chromatographic-tandem mass spectrometric assay was developed and validated to determine the major metabolite of betahistine, 2-pyridylacetic acid, in human plasma.

2.?The analyte was extracted from plasma samples by liquid–liquid extraction and analysed using liquid chromatography-tandem mass spectrometry with an electrospray ionization interface. The method has a lower limit of quantitation of 1?ng?ml^?1 for a 0.5-ml plasma aliquot. The intra- and interday precision (relative standard deviation), calculated from quality control (QC) samples, was less than 10%. Accuracy as determined from QC samples was within ±7%.

3.?The validated method was successfully applied to a pharmacokinetic study of betahistine in healthy volunteers. After oral administration of a single dose of 24?mg betahistine mesylate to 20 healthy Chinese male volunteers, C_max was 339.4?ng?ml^?1 (range 77.3–776.4?ng?ml^?1). The t_1/2 was 5.2?h (range 2.0^?1?11.4?h). The AUC_0?t obtained was 1153.5?ng?ml^?1?h (range 278.5–3150.8?ng ml^?1?h). The disposition of the metabolite exhibited a marked interindividual variation.

4.?The plasma concentrations of the parent drug were less than 0.5?ng ml^?1, suggesting that it undergoes almost complete first-pass metabolism. The reported two active metabolites were not detected in the plasma of any volunteer. Although there is no evidence that the major metabolite has pharmacological activity, the clinical importance of 2-pyridylacetic acid in humans should be reinvestigated.     

Acetylmethadol metabolites influence opiate receptors and adenylate cyclase in amygdala

The activity of acetylmethadol and two major metabolites of this drug, noracetylmethadol and dinoracetyl-methadol were studied in monkey brain amygdaloid tissue. This tissue contains opiate receptors which may be assessed by direct binding studies; also these receptors are coupled to and capable of modulating a dopamine-stimulated adenylate cyclase system. Etrophine and D-ALa2-Met-enkephalin exhibited similar potencies when assessed for inhibition of dopamine-stimulated adenylate cyclase or competition for [3H] D-Ala2-Met-enkephalin binding sites. While acetylmethadol displaced [3H] D-Ala2-Met-enkephalin binding sites with a Ki of 7.4 X 10(-7) M, it had no detectable activity in the opiate receptor coupled adenylate cyclase system. Noracetylmethadol and dinoracetylmethodol, however, were capable of both binding (Ki values, 5.6 and 1140 nM respectively) as well as inhibiting the dopamine stimulated adenylate cyclase system (IC50 values, 1.2 and 800 nM respectively). Thus, it appears that metabolism of acetylmethadol to its mono-demethylated form results in a compound more active in both assay systems. The further demethylation of noracetylmethadol results in a second, but less potent, active metabolite. This process of biological activation can be assumed to account for the slow onset and long duration of action of acetylmethadol.     

Simultaneous determination of glipizide and glimepride by Rp-Hplc in dosage formulations and in human serum

In this article, simple, liquid chromatographic method has been developed and validated for the determination of glipizide and glimepride in pharmaceutical formulations and in human serum. Chromatographic separation was carried out on a Nucleosil, C₁₈ (10?μm, 25?×?0.46?cm) column using the mobile phase 80:20 methanol:water with pH adjusted to 3.5 at a flow rate 1?ml?min^?1. Peak intensity of the drugs was recorded at 230?nm with UV detection. The linearity of the method was studied over the concentration range of 0.15–5?μg?ml^?1 (r?=?0.9979) and 0.5–7.5?μg?ml^?1 (r?=?0.9988) for glipizide and glimepride, respectively. Detection and quantitation limits were found to be 20 and 46?ng?ml^?1 and 70 and 141?ng?ml^?1 for glipizide and glimepride, respectively. There was no significant interference of extra pharmacopeial ingredients and serum observed in the assay of these two drugs.     

Chromatographic determination of drugs of abuse in vitreous humor using solid‐phase extraction

A simple method is presented for the simultaneous determination of morphine, 6‐acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in vitreous humor by high‐performance liquid chromatography with photodiode array detector after solid‐phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250 × 4.6 mm i.d., 5 µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1–4 µg ml^?1, with correlation coefficients higher than 0.99. The limits of detection were lower than 30 ng ml^?1 for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30 µg ml^?1 for morphine, 0.24 µg ml^?1 for 6‐acetylmorphine, 0.10 µg ml^?1 for codeine, 0.81 µg ml^?1 for cocaine, 1.26 µg ml^?1 for benzoylecgonine, 0.15 µg ml^?1 for cocaethylene, 0.11 µg ml^?1 for methadone and 0.68 µg ml^?1 for EDDP were obtained. Copyright © 2012 John Wiley & Sons, Ltd.     

Bioavailability of nifedipine: A comparison between two preparations

In a random cross-over study, eight healthy volunteers received single 10 mg doses of either nifedipine capsule (Adalat, Bayer) or nifedipine tablets (Taro) after an overnight fast. The areas under the serum concentration time curves were not significantly different (AUC^{0→ ∞} 319·8 ± 28·0 (SEM) ng ml^?1 h^?1 for capsules, 260·8 ± 15·3 ng ml^?1 h^?1 for tablets). The peak serum levels and the time of their occurrence were 162·4 ± 23·4 ng ml^?1 at 30 min for capsules and 43·0 ± 3·0 ng ml^?1 at 1–2 h for tablets, indicating that the absorption of nifedipine from the capsule is faster than from the tablet form. Clinical symptoms of vasodilation corresponded with the nifedipine peak levels. We conclude that although the bioavailability in general of the two preparations is similar, the therapeutic equivalence may differ. Depending on the therapeutic indication each preparation may have its merits.     

Inhibition by non-steroidal anti-inflammatory agents of the release of rabbit aorta contracting substance and prostaglandins from chopped guinea-pig lungs

The release of prostaglandins (PGs) and rabbit aorta contracting substance (RCS) was investigated using mechanical agitation of chopped lung tissue from unsensitized guinea-pigs. Manual stirring of the lung tissue for 45 s produced maximal release of prostaglandins, a release corresponding to the effect of [20–40 ng ml^?1 of PGE₂ x 45 s at 5 ml min^?1] about 100 ng PGE₂ on the rat stomach strip. Nonsteroidal anti-inflammatory agents inhibited the release of the contracting substances. Indomethacin, the most potent, was active in concentrations of about 20 ng ml^?1, whereas flufenamic acid was twice, phenylbutazone about 60 times, acetylsalicylic acid 100 times and sodium salicylate about 6000 times less active than indomethacin. The method could prove to be a simple test for screening nonsteroidal anti-inflammatory agents for inhibition of prostaglandin synthesis.     

Identification and quantification of metabolites of arachidonic acid from cultures of endothelial cells by HPLC-MS2

Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) are oxidative products of arachidonic acid, some of which participate in the regulation of vascular tone. Little is known about the production of EETs and HETEs in cultures of endothelial cells. This paper reports an assay for the simultaneous quantification of isomers of EETs and HETEs from endothelial cell culture supernatants by employing solid-phase extraction and liquid chromatography-mass spectrometry. The method enabled measurement of 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 8-HETE, 11-HETE, 12-HETE and 15-HETE. The metabolites were chromatographically separated by reversed-phase HPLC and identified by negative ESI tandem mass spectrometry and this method was used to investigate the metabolism of arachidonic acid with an endothelial cell line. For quantification, the sum of signal intensities of characteristic fragment ions was used. The detection limits for 5,6-EET and of other EET and HETE isomers were 2.0, 0.64 and 8?ng?ml^?1 culture medium, respectively. The precision of the method was determined with spiked culture medium (three concentrations, n?=?5) and the average RSD ranged from 6.0 to 24.2%. The dynamic range was 0.6–23.5?ng?ml^?1 culture medium for EETs and 8.0–200?ng?ml^?1 for HETEs. Arachidonic acid was mainly metabolised to HETEs with product levels ranging from 59.3 to 460?ng 10^?6 cells. The median of 8,9-EET and 14,15-EET was 14.5 and 17.7?ng 10^?6 cells, respectively, whereas 5,6-EET and 11,12-EET were below 2?ng 10^?6 cells in a 5-min incubation assay at a 30?µM arachidonic acid substrate concentration.     

Plasma concentrations and bioavailability of isosorbide dinitrate and pindolol from a combination formulation

The plasma concentrations and bioavailability of sustained-release isosorbide denigrate and standard-release pindolol have been compared after administration of these drugs in combination and alone. Bioavailability parameters of isosorbide dinitrate and pindolol obtained after administration of the drugs in combination were not significantly different (P>0.05) to those obtained after administration of either drug alone. Two peaks of mean concentrations of isosorbide dinitrate occurred in plasma after administration of 30 mg of this drug in combination with 7.5 mg pindolol (4.4 ng ml^?1 at 1 h and 4.5ng ml^?1 at 5h), or alone (5.9ngml^?1 at 2h and 5.7ng ml^?1 at 5h). In each case, plasma concentrations of isosorbide dinitrate were maintained during at least 8 h, whereas the drug was not detected in plasma at 2.5 h after administration of a standardrelease formulation. The peaks of mean concentrations of pindolol were 39.7ng ml^?1 at l.5h after administration of 7.5 mg drug in combination with isosorbide dinitrate and 38.0 ng ml^?1 at 1 h after administration of the drug alone. Concentrations of pindolol in plasma declined with a half-life of 3 h.     

Physiologically based modeling of p‐tert‐octylphenol kinetics following intravenous,oral or subcutaneous exposure in male and female Sprague–Dawley rats

The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model for p‐tert‐octylphenol (OP) for understanding the qualitative and quantitative determinants of its kinetics in Sprague–Dawley rats. Compartments of the PBPK model included the liver, richly perfused tissues, poorly perfused tissues, reproductive tissues, adipose tissue and subcutaneous space, in which OP uptake was described as a blood flow‐ or a membrane diffusion‐limited process. The PBPK model successfully simulated previously published data on blood and tissue OP concentrations in Sprague–Dawley rats following oral, intravenous (i.v.) or subcutaneous (s.c.) routes. The model predicted that OP concentrations would reach 6.8, 13.8 and 27.9 ng ml^?1 (male) and 7.2, 14.7 and 31.4 ng ml^?1 (female), 4 h after a single i.v. dose of 2, 4 and 8 mg kg^?1, respectively. The model also predicted that OP concentrations would reach 53.3, 134.8 and 271.2 ng ml^?1 (male) and 87.4, 221.4 and 449.7 ng ml^?1 (female) 4 h after a single oral dose (50, 125 and 250 mg kg^?1) and that, 4 h after a single s.c. dose (125 mg kg^?1), OP concentrations would reach 111.3 ng ml^?1 (male) and 121.6 ng ml^?1. A marked sex difference was seen in blood and tissue OP concentrations. This was reflected in the model by a gender‐specific maximal velocity of metabolism (V_max) that was higher (1.77×) in male than in female rats. Further studies are required to elucidate the mechanism underlying the gender differences and to evaluate whether that is also observed in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Plasma and cerebrospinal fluid concentrations of pentazocine in patients: assay by mass fragmentography

Mass fragmentography has been used for the determination of low concentrations of pentazocine in blood plasma and cerebrospinal fluid (csf) after intravenous administration of a 30 mg dose to eight patients undergoing neurosurgery under general anaesthesia. A pharmacokinetic analysis based upon mean plasma levels indicated a half-life of 134 min. Lumbar csf levels of pentazocine increased rapidly with mean values from about 3 ng ml^?1 at 5 min to 10 ng ml^?1 at 30 min and to about 15 ng ml^?1 at 90–120 min. The possibility of repeated analyses of drug concentrations in the csf represents an important step towards the correlation of chemical data with clinical effects for centrally acting drugs.     

Pharmacokinetics of isosorbide dinitrate after intravenous infusion in human subjects

Plasma concentrations of isosorbide dinitrate have been measured after intravenous infusion of drug at a rate of 5·0 mg h^?1 for 150 min and after single equal oral doses of 12·5 mg of drug in solution to two normal human subjects. During the infusion, uneven plateau concentrations were approached after 30 min. The calculated average steady-state plasma levels were 258 ng ml^?1 and 514 ng ml^?1 in the two subjects respectively. The half-life of elimination of isosorbide dinitrate after termination of the infusion was 9–10 min. After oral doses, peak plasma levels of 26·6 ng ml^?1 and 12·7 ng ml^?1 occurred at 10 min and 20 min in the two subjects respectively. The terminal half-life of drug after the oral doses was much longer than the elimination half-life (about 10 min), and was associated with the absorption phase. Fairly good agreement was obtained between the observed concentrations and those predicted by a one-compartment open model. The systemic availability of isosorbide dinitrate after the oral doses was up to only 3 per cent of the equal doses infused, indicating that presystemic elimination processes accounted for very large proportions of the oral doses. The systemic clearances of drug after infusion of 0·32 1 min^?1 and 0·161 min^?1 were unexpectedly low for a drug of reported high liver extraction ratio.     

Assessment of the value of therapeutic monitoring of tacrine in Alzheimer's disease

Objectives: The purpose of this study was to validate the lower end of the putative therapeutic range of serum tacrine concentrations of 7–20?ng?·?ml^?1 in the treatment of Alzheimer's disease. Methods: The relationship between dose, steady-state serum tacrine concentrations and change in MMSE score (a measure of cognitive function) was examined in 106 Alzheimer's disease patients who had been treated with the drug for 12 weeks. Results: In all, 72% of patients showed some response, but there was no relationship between dose and the chance of a favourable outcome. The proportion of patients with serum concentrations above 7?ng?·?ml^?1 who improved (79%) was significantly greater than that of those with serum concentrations below this level (47%) (P?<?0.02). Also, a significantly greater proportion of patients with serum concentrations above both 5?ng?·?ml^?1 and 9?ng?·?ml^?1 showed improvement in comparison to those with concentrations below these levels. Conclusions: This study indicates that therapeutic monitoring of serum tacrine concentrations might increase the possibility of responding to tacrine by some 68%. This represents an important contribution to the management of Alzheimer's disease patients with this drug, and may also be relevant to the use of the newer generation of cholinesterase inhibitors.     

Quantitation of iprindole in plasma by GLC

A specific, sensitive, rapid, and reproducible method for iprindole in human plasma was developed using gas chromatography with trimipramine as internal standard. The sensitivity of the method is of S ng ml^?1 and a linearity was obtained for concentrations ranging from 12.5ngml^?1 to 100ngml^?1 with a regression coefficient of 0.9872. The plasma levels of iprindole were determined in five healthy volunteers following the administration of a single oral dose of 60 mg and in four patients admitted for endogenous depression after a 3-week administration of 90mgd^?1. Following a single oral dose to healthy volunteers, the maximum concentrations occurred between 2 and 4h after administration of the drug and the mean half-life value was 52.5 h. In patients the steady-state concentrations of iprindole ranged between 18 and 77ngml^?1.     

Methadone radioimmunoassay: two simple methods

Two simple and economical radioimmunoassays for methadone in blood or urine are described. Haemolysis, decomposition, common anticoagulants and sodium fluoride do not affect the results. One assay uses commercially-available [1^?3H](-)-methadone hydrobromide as the label, while the other uses a radioiodinated conjugate of 4-dimethylamino-2,2-diphenylpentanoic acid and L-tyrosine methyl ester. A commercially-available antiserum is used in both assays. Normethadone and α-methadol cross-react to a small extent with the antiserum while methadone metabolites, dextropropoxyphene, dipipanone and phenadoxone have negligible cross-reactivities. The ‘cut-offs’ of the two assays as described are 30 and 33 ng ml^?1 for blood, and 24 and 21 ng ml^?1 for urine. The assay using the radioiodinated conjugate can be made more sensitive if required by increasing the specific activity of the label.     

Determination,pharmacokinetics and protein binding of a novel tissue-type plasminogen activator,pamiteplase in human plasma

1. An enzyme-linked immunosorbent assay (ELISA) and functional bioassay to determine immunoreactive and bioactive concentrations of pamiteplase, a novel thrombolytic agent, in human plasma were developed. The ELISA and functional bioassay showed satisfactory accuracy and precision within a concentration range of 0.5-25 ng.ml^?1 and of 0.127-16.2 ng.ml^?1 respectively. 2. The pharmacokinetics of pamiteplase in healthy human subjects were evaluated using the ELISA and functional bioassay. Irrespective of the method used, plasma concentrations declined bi-exponentially. Half-lives in the β phase were 1.25 and 0.78 h, and AUCs were 507.9 and 286.4 ng.h.ml^?1 respectively. Total clearances of pamiteplase decreased to 19 and 31% of those of the wild-type tissue-type plasminogen activator. 3. The protein binding of pamiteplase in human plasma was investigated by gel filtration chromatography. Pamiteplase formed three high molecular weight complexes with α₂-macroglobulin, C₁-esterase inhibitor and α₂-plasmin inhibitor in human plasma. This complex formation was relatively slow, and was thought to be irreversible and covalently bounded. Furthermore, this protein binding in humans resulted in the termination of biological action.     

The development of a sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A receptor antagonist, in human plasma

A sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A (CCK-A) receptor antagonist, was developed to study the pharmacokinetics of the drug at low-dose administration using a specific monoclonal antibody. The high performance liquid chromatography (HPLC) method had been used for studying toxicokinetics, but its determination limit (2.5 ng ml⁻¹) was too high for use in clinical studies. Subsequently we developed an enzyme immunoassay (EIA) using rabbit anti-FK480 serum (polyclonal antibody). It had higher sensitivity (0.1 ng ml⁻¹) when 0.5 ml of plasma was used but its specificity was low because of the cross-reactivity of the metabolites of FK480. Therefore we produced several monoclonal antibodies for FK480 by cell fusion, and selected the antibody which was least cross-reactive for the isolated metabolites of FK480. Finally we developed a sensitive and specific EIA using this monoclonal antibody. The lower limit of quantification of this method was 0.2 ng ml⁻¹ when 0.2 ml of human plasma was used. The coefficient of variation over the calibration range (0.2–10 ng ml⁻¹) was less than 15%. We used this method for clinical studies, and it showed a good correlation to the HPLC method when plasma concentration was 2.5 ng ml⁻¹ or more.     

Determination of fentanyl, metabolite and analogs in urine by GC/MS

A rapid and sensitive method for the simultaneous determination of alfentanyl, sufentanyl and fentanyl (and its major metabolite norfentanyl) in urine was developed and validated. The method involved a liquid–liquid extraction in alkaline conditions, derivatization with pentafluoropropionic anhydride to improve the sensitivity for norfentanyl and subsequent analysis in GC/MS. The LODs are 0.08 ng ml^?1 for all substances (0.04 ng ml^?1 for alfentanyl). Intra‐ and inter‐day precision coefficient of variation was always below 15%; mean relative error (accuracy) was always below 15%. The method was linear for all analytes, with quadratic regression of calibration curves always higher than 0.99. The method was applied to real samples of subjects who had received therapeutic doses of fentanyl, showing its suitability for the determination of low levels of these substances. The method was also applied to a subject whose death was attributed to fentanyl overdose. Copyright © 2010 John Wiley & Sons, Ltd.     

Disposition of LY333531, a selective protein kinase C β inhibitor,in the Fischer 344 rat and beagle dog

1. Studies were conducted in the Fischer 344 rat and beagle dog to determine the disposition of LY333531 and its equipotent active des-methyl metabolite, LY338522, both potent and selective inhibitors of the β-isozyme of protein kinase C. 2. Male Fischer 344 rats and female beagle dogs received a single 5-mgkg^?1 oral dose of ¹⁴C-LY333531. Urine, faeces, bile and plasma were collected and analysed for ¹⁴C, LY333531 and LY338522. 3. LY333531 was eliminated primarily in the faeces (91% by 120 h in rat, 90% by 96h in dog). Bile contributed the majority of the radioactivity excreted in the faeces in rat (66% in the cannulated bile duct study) and a variable but significant proportion in dog. 4. Pharmacokinetics following a single 5?mg kg^?1 oral dose of ¹⁴C-LY333531 to the male rat produced C_max and AUC_0-∞ for LY333531 of 14.7 ng ml^?1 and 60.8ng h ml^?1, respectively, with a half-life of 2.5 h. LY338522 and total radioactivity showed similar profiles. 5. In the female dog at the same dose, C_max and AUC_0-∞ of LY333531 were higher, producing 245 ± 94 ng ml^?1 and 1419 ± 463ng h ml^?1, respectively, with a half-life of 5.7 h. 6. The data indicate that the disposition of LY333531 is similar in rat and dog.     

Two metabolites of sulphinpyrazone and their identification and determination by mass spectrometry

Sulphinpyrazone is an antiplatelet agent in vivo and in vitro. Two active metabolites, a sulphide (S) and a hydroxylated sulphide (S-OH) have been identified in rabbit and human plasma and a selective and sensitive g.c.-m.s.-method for quantitative determination of the sulphide and hydroxylated sulphide in plasma and urine has been evolved which allows concentrations down to 5 ng ml^?1 for the sulphide and 30 ng ml^?1 for the hydroxylated sulphide to be detected. The time course of the metabolite concentrations in plasma corresponds to the biological findings, suggesting that the metabolites contribute significantly to the in vivo effects of the drug.