Ochratoxin A in the morning and afternoon portions of urine from Coimbra and Valencian populations.

The widespread contamination of foodstuffs and beverages by mycotoxins, such as ochratoxin A (OTA), has made the monitoring of human contamination levels essential. By using a sensitive, accurate and speedy method that combines extraction with 5% NaHCO(3), immunoaffinity column clean-up and HPLC with fluorescence detection, the human exposure to OTA through urine analysis can be monitored. This method is less invasive than blood monitoring and has the potential to be a good marker of human exposure. The limit of quantification of the method was 0.007 ng/mL of urine, with recoveries of OTA, from urine samples spiked at levels between 0.02 and 0.1 ng/mL, higher than 91% with RSD lower than 15.5%. This study evaluated OTA contamination levels in human urine sample fractions, collected in the morning and afternoon, in two populations, one from Coimbra city, in Portugal, and another from the Valencian community, in Spain. In the Coimbra population, 60 samples from 30 healthy individuals were analyzed, levels of OTA in 13 morning samples and 14 afternoon samples having been detected, with concentrations ranging from 0.011 to 0.208 and 0.008 to 0.11 ng/mL respectively. In the Valencia population, 62 samples from 31 healthy individuals were analyzed, with OTA being detected in 25 morning samples and 26 afternoon samples. The concentrations varied between 0.007 and 0.124 ng/mL in the morning samples, and 0.008 and 0.089 ng/mL in the afternoon samples. Significant differences were found between the morning levels of OTA from both populations (P=0.033). For afternoon samples, significant differences were not found, P value=0.163.

Development and validation of two LC-MS/MS methods to assay urinary tylerdipine hydrochloride and its metabolites in healthy Chinese subjects.

For quantitative assaying tylerdipine hydrochloride, and its two primary metabolites (M2 and M4) in human urine, two sensitive and accurate LC-MS/MS methods were firstly developed and validated, where multiple reaction monitoring (MRM) was applied under positive electrospray ionization mode for tylerdipine and negative electrospray ionization mode for M2/M4, respectively. Urinary proteins were precipitated using acetonitrile, and deuterated isotopes of tylerdipine and M4 ([D5]­tylerdipine and [D6]-M4) were used as internal standards. Triton X-100, a good surfactant, was used to prevent the adsorption. An Agilent Poroshell 120 column was employed for chromatographic separation of the analytes with the mobile phases of 2 mM ammonium formate solution (containing 0.1% formic acid) and acetonitrile (45:55 for tylerdipine and 75:25 for the M2/M4, v/v). Flow rate was 0.3 mL/min. Calibration curves for tylerdipine, M2 and M4 in urine were linear over the ranges of 0.02-10 ng/mL, 2-1500 ng/mL and 0.5-200 ng/mL, respectively. The precision, accuracy, specificity and stability of two methods all evaluated and achieved the acceptable criteria. The LC-MS/MS methods were successfully applied to assay urinary excretion of tylerdipine and the metabolites in healthy Chinese subjects who orally received a single dose of 20 mg tylerdipine tablet. Generally, the urinary excretion of the two primary metabolites accounted for 11.7% of the total dose of tylerdipine in healthy Chinese subjects, while little tylerdipine was recovered in urine.

Lomerizine, trimetazidine and bis-(4-fluorophenyl)-methylpiperazine in human urine after oral administration of lomerizine dihydrochloride: analysis by liquid chromatography-high resolution-tandem mass spectrometry.

In sports drugs testing, the differentiation between the abuse of the prohibited substance trimetazidine and that of the permitted drug lomerizine is required because trimetazidine is one of the metabolites of lomerizine. Therefore, it is important to identify a lomerizine-specific metabolite in urine that allows making the distinction. In this study, a simple dilute-and-shoot method employing liquid chromatography-high resolution-tandem mass spectrometry for the quantification of trimetazidine, lomerizine and the specific metabolite bis-(4-fluorophenyl)-methylpiperazine (M6) in urine was developed. An oral dose of 15 mg was administered to 10 male volunteers, after which urine samples collected during the following 276 hours were analyzed using the developed method, allowing for examination of the target analytes' excretion profile. The limit of detection of all target analytes was <0.02 ng/mL. In all volunteers, the metabolite M6 was detected up to 276 hours after administration. After more than 12 hours, all volunteers were found to have higher concentrations of the metabolite M6 than of trimetazidine. The concentrations of trimetazidine, lomerizine, M6, and the M6/trimetazidine ratio in the final sample collected after 276 hours were 0.2-0.9 ng/mL, <0.05-0.1 ng/mL, 14.1-38.3 ng/mL, and 28.8-122.9, respectively. The urinary excretion of trimetazidine, unchanged lomerizine, and the metabolite M6 within the first 276 hours was 0.64%, 0.006%, and 6.1%, respectively. Consequently, the absence of the metabolite M6 in doping control urine samples corroborates the conclusion that lomerizine is unlikely to be the source of trimetazidine. The results confirm that the M6 metabolite is the longest-lasting urinary metabolite of lomerizine currently known.

Two cases of fatal amlodipine overdose.

Two fatal overdoses of the calcium channel blocker amlodipine are described. Postmortem samples were screened for volatiles and therapeutic and abused drugs. Amlodipine was measured by liquid chromatography-atmospheric pressure photoionization-mass spectrometry. The heart blood amlodipine concentrations for the two cases were 2.4 and 0.95 mg/L, and amlodipine was quantified in all other tissues. In the first case, venlafaxine and norvenlafaxine were also found, and the angiotensin receptor antagonist olmesartan was tentatively identified. The concentrations of amlodipine are compared with previously reported fatal and nonfatal overdoses. The medical examiners ruled in both cases that the manner of death was suicide and the causes of death were mixed drug intoxication and amlodipine intoxication.

Analytical detection of trimetazidine produced by metabolic conversion of lomerizine in doping control analysis.

The identification of trimetazidine in urine samples might result from administration of the permitted drug lomerizine. Laboratories are therefore urged to carefully investigate suspicious cases where trimetazidine is detected. Differentiation of abuse of the banned substance trimetazidine from use of the permitted drug lomerizine would be supported by analysis of the intact drug lomerizine and/or specific metabolites. Copyright © 2015 John Wiley & Sons, Ltd.

Pharmacokinetic interaction between verapamil and almotriptan in healthy volunteers.

OBJECTIVE: To assess the interaction between almotriptan, a 5-HT1B/1D-receptor agonist used to treat migraine, and verapamil, an agent for migraine prophylaxis. METHODS: Twelve healthy volunteers received the following treatments in a crossover design: (1) 120-mg sustained-release verapamil tablet twice daily for 7 days and one 12.5-mg almotriptan tablet on day 7 and (2) one 12.5-mg almotriptan tablet alone on day 7. Serial plasma and urine samples were obtained on day 7. Almotriptan plasma concentrations were determined by liquid chromatography-tandem mass spectrometry; urine samples were analyzed by ultraviolet HPLC. Safety measures included blood pressure and pulse measurements, electrocardiography, and adverse event monitoring. Statistical comparisons of pharmacokinetic parameters and vital sign data were made by ANOVA. RESULTS: Mean almotriptan peak concentration and area under the plasma concentration-time curve were significantly higher and volume of distribution and oral clearance were significantly lower after coadministration of almotriptan and verapamil compared with administration of almotriptan alone. The magnitudes of these differences were approximately 20%. Renal clearance was unaffected by verapamil coadministration. No significant effects of treatment on blood pressure or pulse were detected, with the exception of sitting systolic blood pressure at 2 hours after administration. However, the difference in mean change from baseline at this time point was only 8 mm Hg. CONCLUSIONS: Verapamil modestly inhibited almotriptan clearance to a degree consistent with the modest contribution of CYP3A4 to almotriptan metabolism. This observation and the lack of effect of verapamil on the tolerability to almotriptan administration suggest that no reduction of the almotriptan dose is warranted.

Determination of the dihydropyridine aryloxypropanolamine Z6568 and its acidic metabolites in plasma, urine and tissues by solid phase extraction and liquid chromatography/negative-ion mass spectrometry.

A sensitive and selective liquid chromatographic/mass spectrometric assay for the determination of the dihydropyridine aryloxypropanolamine compound Z6568 and its monoacidic metabolites in plasma, urine and tissues has been developed and validated. Z6568 and the acidic metabolites were isolated from the biological matrices by solid-phase extraction, separated by reversed-phase high-performance liquid chromatography and detected by mass spectrometry using a thermospray interface. Filament ionization and negative-ion detection were used, monitoring the molecular ion of Z6568 and a major fragment of the acidic metabolites. The analysis of spike plasma, urine and tissues demonstrated good accuracy and precision of the assay with a detection limit of 0.5 ng ml-1 for Z6568 and 2 ng ml-1 for the metabolites. Data relative to the optimization of the extraction procedure, mass spectrometric conditions and the validation protocol are reported. The method was used to investigate the metabolic fate and tissue distribution in laboratory animals.

Bioavailability and disposition of terodiline in man.

Terodiline was concomitantly administered intravenously (12.5 mg) and orally ([2H]terodiline, 12.5 mg) to 10 healthy volunteers. In four of the subjects, a tracer dose of the intravenously given terodiline was 3H-labeled. In a separate study, six subjects were given [3H]terodiline orally. Estimated pharmacokinetic parameters were as follows: systemic clearance, 93 mL/min; renal clearance, 14 mL/min; volume of distribution at steady-state, 407 L; terminal half-life, 54 h; and mean residence time, 77 h. After intravenous infusion, a rapid distribution phase (half-life, 4.5 min) could be observed. The maximum serum concentration after the oral dose was 29 micrograms/L and the time to maximum concentration was 5 h (estimated by noncompartmental analysis). Absorption commenced within the first hour and by deconvolution the maximum rate of absorption was determined to occur between 1 and 3 h, and by 3.4 h 90% of the available dose had been absorbed. Calculation of bioavailability by noncompartmental AUC, two-compartmental analysis, urinary excretion, and 24-h oral/intravenous concentration ratio gave similar results (ANOVA test, not significant). About 75% and 25% of administered radioactivity could be recovered in urine and feces, respectively. Intact terodiline in feces accounted for about 1% of the dose. p-Hydroxyterodiline was quantitated in feces and accounted for about 5% of the dose. Another metabolite, 3,4-dihydroxyterodiline, which has not previously been detected in urine or serum, was also identified.

Determination of lacidipine from urine by HPTLC using off-line SPE.

A simple, rapid and sensitive high performance thin layer chromatographic method was developed and validated for the estimation of lacidipine. The sample preparation involved protein precipitation followed by an efficient solid phase extraction on C18 cartridge. The analytes were isolated from 1 ml of urine and recovered by pure ethyl acetate solution. The method employed TLC aluminium plate precoated with silica gel 60F254 as the stationary phase. The solvent system employed consists of toulene-ethyl acetate [6.5:3.5v/v]. This system gave a dense and compact spot of the drug at R(f) value of 0.45. The linear regression data for the calibration plots showed good linear relationship (r=0.999) over the concentration range 10-80 ng. Recovery studies were performed at two different levels. The recovery data reveals that the R.S.D. for intra-day and inter-day analysis at 10 ng was found to be 0.84 and 0.22%, respectively. The proposed method was found to be useful for the routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Voltammetric determination of isradipine in dosage forms and spiked human plasma and urine.

The voltammetric behavior of isradipine was studied using direct current (DC(t)), differential-pulse (DPP) and alternating current (AC(t)) polarography. Isradipine exhibited well-defined cathodic waves over the whole pH range in Britton-Robinson buffer (BRb). At pH 5, the analytical pH, the diffusion-current constant (Id) was 8.27+/-0.52. The current-concentration plots were rectilinear over the range 1-20 and 0.1-18 microg/ml using the DC(t) and DPP modes, respectively, with minimum detectability of 0.01 microg/ml (2.7 x 10(-8) M) using the latter technique. The current has been characterized as being diffusion-controlled, although adsorption phenomenon played a limited role in the electrode process. The proposed method was applied to commercial tablets and capsules. The percentage recoveries were in good agreement with those given by the manufacturer. The method was further extended to the in-vitro determination of the drug in spiked human urine and plasma, the percentage recoveries were (n = 4) 100.12+/-1.42 and 103.88+/-5.13, respectively. The number of electrons involved in the reduction process was accomplished and a proposal of the electrode reaction was presented.

Screening procedure for detection of dihydropyridine calcium channel blocker metabolites in urine as part of a systematic toxicological analysis procedure for acidic compounds by gas chromatography-mass spectrometry after extractive methylation.

A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for the detection of dihydropyridine calcium channel blocker ("calcium antagonist") metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 139, 284, 297, 298, 310, 312, 313, 318, 324, and 332, the possible presence of calcium channel blocker metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of amlodipine, felodipine, isradipine, nifedipine, nilvadipine, nimodipine, nisoldipine, and nitrendipine in human urine samples. Because urine samples from patients treated with nicardipine were not available, the detection of nicardipine in rat urine was studied. The overall recovery ranged between 67 and 77% with a coefficient of variation of less than 10%, and the limit of detection was at least 10 ng/mL (signal-to-noise ratio = 3) in the full-scan mode.

Diltiazem and pentoxifylline determination in postmortem specimens.

A 78-year-old woman was found dead in her basement. Qualitative screening of available postmortem specimens detected the presence of diltiazem and pentoxifylline. Quantitations were carried out by gas chromatography using nitrogen-phosphorus detection and confirmed by gas chromatography-mass spectrometry with the following results: blood, 0.59 mg/dL diltiazem and 0.63 mg/dL pentoxifylline; urine, 1.17 mg/dL diltiazem and 0.08 mg/dL pentoxifylline; bile, 0.40 mg/dL diltiazem and 0.22 mg/dL pentoxifylline; gastric contents, 0.28 mg/dL diltiazem and 0.02 mg/dL pentoxifylline. Both drugs were found qualitatively in formaline-fixed tissues.

Spectrofluorometric determination of nimodipine in dosage forms and human urine.

A simple sensitive and specific spectrofluorometric method was developed for the determination of nimodipine (NDP) in pharmaceutical preparations and human urine. The method is based on reduction of nimodipine with Zn/HCl and measuring the obtained fluorescence at 425 nm after excitation at 360 nm. The factors affecting the development of the fluorophore and its stability were studied and optimized. The effect of some surfactants such as beta-cyclodextrin (betaCD), carboxymethylcelullose (CMC), sodium dodecyl sulphate (SDS) and Triton X-100, on the fluorescence intensity was studied. The fluorescence intensity-concentration plot is rectilinear over the range 0.1-5.0 microg/ml in presence of Triton X-100 with a minimum detectability limit of 0.06 microg/ml (1.62 x 10(-7) M). The proposed method was successfully applied to commercial tablets containing NDP, the percentage recovery agreed well with those obtained using the official methods. The method was further extended to the in vitro determination of NDP in spiked human urine samples. The % recovery was 102.1 +/- 2.54 (n = 4). A proposal of the reduction reaction pathway was postulated.

Urinary excretion of the 1,4-dihydropyridine calcium antagonist VULM 993 and its metabolites in the rat.

After oral dose of the 1,4-dihydropyridine calcium antagonist 14C-VULM 993 (50 mg/kg) a mean of 44.5% of the administered radioactivity was excreted via urine during the first 72 hours. Using an extractive fractionation procedure, the urinary metabolites were classified on the basis of their polarity and acidic/basic properties. Approx. 40% of total urine metabolites were found to be polar, non-extractable compounds--mostly glucuronide/sulphate conjucates. About one half of all urine metabolites were shown to possess overall acidic nature. G.l.c.-m.s. and t.l.c.-m.s. analyses of urine extracts revealed the presence of only minor amounts of the parent drug toghether with six metabolites identified as products of 1,4-dioxaspiro[4,4]nonane moiety cleavage, hydrolysis of one or both ester side functions also combined with 1,4-dihydropiridine nucleus dehydrogenation. Technique of thin-layer radio-chromatography was used to quantify urinary excretion rates of the parent drug and the established metabolites.

Circadian variations in the pharmacokinetics, tissue distribution and urinary excretion of nifedipine after a single oral administration to rats.

Circadian variations in the pharmacokinetics, tissue distribution and urinary excretion of nifedipine were examined in fasted rats after administering a single oral dose at three different dosing times (08:00 am, 16:00 pm, 00:00 am). The plasma concentrations, the areas under the plasma concentration-time curve from zero to 6 h (AUC(0-6 h)) and the peak plasma concentration (C(max)) were significantly higher in the rats dosed at 08:00 am (immediately inactive), and was lower at 16:00 pm (most inactive) and 00:00 am (most active). The time to reach the C(max) (T(max)) was the shortest in the rats dosed at 08:00 am. It was very interesting to observe the double peak phenomena in the plasma concentration profiles, showing a larger peak followed by a smaller peak. There was a dosing time dependency on the tissue distribution 30 min after administration, showing a similar tendency to the pharmacokinetic behavior. However, there was no distinct dosing time dependency observed at 2 h after administration due to the extensive disposition. The cumulative urine excretion of nifedipine in the rats dosed at 08:00 am was significantly higher (about two-fold) than in those dosed at 16:00 pm and 00:00 am. The pharmacokinetics of nifedipine in the rats was consistent with that observed in human subjects in terms of the day-night clock time but the biological time was the opposite, as marked by the rest-activity cycles. These results may help to explain the circadian time-dependency of nifedipine pharmacokinetics.

Direct determination of naftopidil by non-protected fluid room temperature phosphorescence.

A selective and sensitive room temperature phosphorimetric method for the direct determination of naftopidil in biological fluids is described. The method is based on obtaining a phosphorescence signal from this antihypertensive drug using TlNO3 as a heavy atom perturber and Na2SO3 as a deoxygenator agent without a protective medium. This technique is named non-protected room temperature phosphorescence (NP-RTP), and enables us to determine analytes in complex matrices without the need for a tedious prior separation process. The optimization of Na2SO3 (8.5 x 10(-3) M) and the accurate value of pH (9.0) were determined using a simplex as a method of optimization. Sodium carbonate-hydrogencarbonate buffer solution (5.0 x 10(-2) M) was used to adjust the suitable pH. The optimum concentration of Tl+ (8.5 x 10(-2) M) was also determined. The delay time, gate time and time between flashes selected were 200 microseconds, 200 microseconds and 5 ms, respectively. Under the above conditions we propose a method to determine naftopidil by direct measurement of phosphorescence intensity with an emission wavelength of 526 nm and an excitation wavelength of 296 nm in the concentration range 0.05-1.00 mg L-1. Under these conditions the phosphorescence signal appears in 3 min once the sample has been prepared. Optimization of the various conditions permitted the establishment of an NP-RTP method for the determination with a detection limit, according to the error propagation theory, of 21.0 ng mL-1. The repeatability was studied using 10 solutions of 0.20 mg L-1 of naftopidil; if error propagation is assumed, the relative error is 1.39%. The standard deviation for replicate samples was 1.1 x 10(-2) mg L-1. This method was successfully applied to the determination of naftopidil, in human urine with recoveries between 106 and 112%.

Solid-phase extraction-high-performance liquid chromatography determination of verapamil and norverapamil enantiomers in urine.

A simple, rapid, sensitive and selective method has been developed for the stereospecific determination of verapamil and its metabolite, norverapamil in urine. For sample preparation we utilized a membrane-based solid-phase extraction (SPE) disk consisting of a thin, particle-loaded membrane inserted in a plastic syringe-like barrel. The particles, which may be C8 or C18 bonded phase (C8 in this work), are embedded within a matrix of PTFE (Teflon) fibrils. Overall analyte recoveries were above 85%, even at low concentration of 3.0 ng/ml with reproducibilities (C.V. values) below 13.1%. This method of extraction has the advantage of speed and considerable reduction in solvent volumes compared to conventional SPE and solvent extraction. The separation of all the enantiomers was achieved using a single chiral stationary phase column, the cellulose-based reversed-phase, Chiralcel OD-R. Analyte concentrations of less than 3.0 ng/ml could be quantitated with C.V. values below 14%. Calibration curves were linear in the range 2.5-300 ng/ml. Intra-day and inter-day reproducibilities were 10.5-14.2% at 3 ng/ml, 4.8-9.3% at 138.5 ng/ml and 7.8-10.1% at 280 ng/ml level, respectively, for all the enantiomers.

Metabolic fate of a new dihydropyridine calcium antagonist, CD-349, in rat and dog.

1. The metabolic fate of 14C-CD-349, a new calcium antagonist, was studied in rat and dog. 2. After oral administration of 14C-labelled drug in both species, the plasma levels of radioactivity reached maxima at 1-2 h and declined with elimination half-lives of 6-7 h. In both species, 71-85% of radioactivity was excreted in faeces and 17-27% in urine in 120 h. Biliary excretion in rat after oral doses amounted to 33%. 3. The low ratio of unchanged drug to total radioactivity in plasma suggested that CD-349 underwent rapid metabolism in both species. 4. Twenty-two metabolites were isolated and identified from dog urine and an incubation mixture with 9000 g rat liver supernatant. Principal routes of biotransformation of CD-349 were similar in both species, and involved: (1) oxidation of the dihydropyridine ring to the corresponding pyridine ring; (2) denitration of the nitrate ester; (3) hydrolysis of the carboxy ester to the carboxylic acid; and/or (4) oxidation of the side chain, although quantitative interspecies differences were observed.

Verapamil: identification of novel metabolites in cultures of primary human hepatocytes and human urine by LC-MS(n) and LC-NMR.

1. Verapamil is a well-known and world-wide prescribed calcium antagonist, but it suffers from extensive first-pass metabolism. Although it has been marketed for many years, a complete understanding of its biotransformation in humans is still lacking. 2. The metabolism of verapamil was therefore investigated in cultures of primary human hepatocytes and in extracts of human urine after oral dosing. Identification of metabolites was done with LC-MS(n) and LC-NMR (600 MHz) to obtain in-depth information on its biotransformation products and definitive proof of the proposed chemical structures of metabolites. 3. Hyphenation of LC-MS(n) and LC-NMR was shown to be a powerful and effective platform for the identification of metabolites. Indeed, 21 Phase I and 16 Phase II metabolites were identified. Basically, all the Phase II metabolites (glucuronides) and 11 of the Phase I (oxidative) metabolites were not reported previously. 4. New insight into verapamil's biotransformation pathway is provided as well as evidence about its true complexity of metabolic disposal.

The metabolism of nicardipine hydrochloride in healthy male volunteers.

Four human volunteers given a 30 mg oral dose of nicardipine hydrochloride containing 40 microCi of the 14C-labelled material achieved peak plasma levels of compound-related radioactivity within one hour of dosing. Parent compound comprised only a minor fraction of the circulating radioactivity indicating rapid first-pass metabolism. Plasma radioactivity declined to background levels within 96 h and was excreted both in the urine and faeces. Urinary excretion was the favoured route comprising about 60% of the dosed radioactivity. Mean total recovered radioactivity amounted to 94.8%. Both 1,4-dihydropyridine and pyridine metabolites of nicardipine hydrochloride were excreted in the urine. The major urinary metabolites, comprising some 36% of the radioactivity excreted in the 0-8 h post-dose period, were the glucuronide conjugates of +/- 2-hydroxyethyl methyl-1,4-dihydro-2,6-dimethyl-4(m-nitrophenyl)-3,5-pyridine dicarboxylate and its pyridine from 2-hydroxyethyl methyl-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridine dicarboxylate.