Matrix Effect Compensation in Small-Molecule Profiling for an LC-TOF Platform Using Multicomponent Postcolumn Infusion.

The possible presence of matrix effect is one of the main concerns in liquid chromatography-mass spectrometry (LC-MS)-driven bioanalysis due to its impact on the reliability of the obtained quantitative results. Here we propose an approach to correct for the matrix effect in LC-MS with electrospray ionization using postcolumn infusion of eight internal standards (PCI-IS). We applied this approach to a generic ultraperformance liquid chromatography-time-of-flight (UHPLC-TOF) platform developed for small-molecule profiling with a main focus on drugs. Different urine samples were spiked with 19 drugs with different physicochemical properties and analyzed in order to study matrix effect (in absolute and relative terms). Furthermore, calibration curves for each analyte were constructed and quality control samples at different concentration levels were analyzed to check the applicability of this approach in quantitative analysis. The matrix effect profiles of the PCI-ISs were different: this confirms that the matrix effect is compound-dependent, and therefore the most suitable PCI-IS has to be chosen for each analyte. Chromatograms were reconstructed using analyte and PCI-IS responses, which were used to develop an optimized method which compensates for variation in ionization efficiency. The approach presented here improved the results in terms of matrix effect dramatically. Furthermore, calibration curves of higher quality are obtained, dynamic range is enhanced, and accuracy and precision of QC samples is increased. The use of PCI-ISs is a very promising step toward an analytical platform free of matrix effect, which can make LC-MS analysis even more successful, adding a higher reliability in quantification to its intrinsic high sensitivity and selectivity.

In vivo oxidative cleavage of a pyridine-carboxylic acid ester metabolite of nifedipine.

The pharmacokinetics of the primary pyridine metabolite of nifedipine (2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinecarboxylic acid dimethylester) (M-0) and its [2H6]dimethylester analog ([2H6]M-0) were studied in male rats. A large, 5.8-fold deuterium isotope effect for the formation clearance of the monomethylester (M-1) was observed, which is strongly indicative for an oxidative reaction mechanism involving the abstraction of a hydrogen atom, presumably by cytochrome P-450. M-0 exhibited a high systemic blood clearance (104 +/- 27 ml/min/kg) (mean +/- SD) which was not significantly influenced by deuterium substitution (125 +/- 13 ml/min/kg). Its systemic clearance is presumably flow limited, and extrahepatic metabolism can be anticipated. The major metabolic pathway for M-0 in male rats seems to be a direct oxidation at the 2-methyl position and subsequently a rapid conversion of the unstable 2-hydroxymethyl-dimethylester to the lactone of the monomethylester (M-2), as has been shown by others in vitro. Non-oxidative ester cleavage of M-0 in our rats was negligible. Deuterium substitution of M-0 at the ester methyl groups induced "metabolic switching" in favor of the direct oxidation of M-0 to M-2.

Voltammetric determination of nilvadipine in dosage forms and spiked human urine.

The voltammetric behaviour of nilvadipine was studied adopting direct-current, differential-pulse and alternating current polarography. Nilvadipine-being nitroderivative-exhibited well-defined cathodic waves over the whole pH range in Britton-Robinson buffers. At pH 5, the diffusion-current constant, (Id) was 4.78. The current-concentration plots are rectilinear over the range 1.5-20 and 0.2-10 microg/ml using the direct current and differential pulse-polarographic techniques with minimum detectability of 0.05 microg/ml (1.3 x 10(-7) M) using the latter technique. The proposed method was applied to commercial capsules containing the drug. The percentage recoveries were in agreement with those obtained by a reference method. Furthermore, the method was applied to spiked human urine, the percentage recovery was 95.54+/-2.137.

Nifedipine concentrations in maternal and umbilical serum, amniotic fluid, breast milk and urine of mothers and offspring.

Eleven hypertensive pregnant patients were treated with nifedipine orally 10 mg thrice daily. At steady state, nifedipine concentration, measured by the high-performance liquid chromatographic method, was 4.3 +/- 1.0 ng/ml (mean +/- s.e.m.) in maternal serum in the third trimester of pregnancy. During delivery, the parent drug levels in maternal and umbilical serum and in amniotic fluid were 12.4 +/- 4.0, 8.6 +/- 2.3 and 2.5 +/- 1.2 ng/ml, respectively. Small amounts of nifedipine were found also in urine of the neonates. In breast milk, the nifedipine concentration was 4.1 +/- 0.8 ng/ml on the third day after delivery. The antihypertensive effect of nifedipine did not correlate with the serum drug concentration. The outcome of pregnancy was favourable in all cases.

Lack of mutagenicity of nifedipine: a possible metabolic implication.

Nifedipine, being a nitro aromatic compound, is capable of undergoing reduction via hydroxyl-amino intermediate to an amine. Such an intermediate is a mutagenic culprit. The urinary metabolites of nifedipine were investigated in order to allay the fear of the existence of this metabolic route in humans. Nifedipine (20 mg) was administered twice daily for 2 weeks. No nitro-reduction product was detected over a 1-month period. Nifedipine lacks mutagenicity in the absence or presence of drug metabolizing microsomes in Salmonella typhimurium TA 98 and Salmonella typhimurium TA 100.

Photostability studies for micellar liquid chromatographic determination of nifedipine in serum and urine samples.

Nifedipine is a photosensitive compound that is converted into its 4-(2-nitrophenyl) pyridine and 4-(2-nitrosophenyl) pyridine homologue. In order to obtain the most adequate conditions for handling nifedipine solutions in the analytical laboratory, a number of studies on the decomposition of this compound were performed. A simple micellar liquid chromatographic procedure was described to determine nifedipine in different biological matrices such as serum and urine, and to control its decomposition. To perform the analysis, nifedipine was dissolved in 0.1 m SDS at pH 3 and chromatographed using a mobile phase containing 0.125 m SDS-3% pentanol, pH 3 on a C18 column and UV detection at 235 nm. The chromatographic analysis time was 8 min. The response of the drug for both biological matrices was linear in the 1-100 microg/mL range, with r2>0.997 at all times. Repeatability, intermediate precision (CV, %) and limits of quantification and detection (ng/mL) were 0.19, 4.3, 104 and 31 in serum and 0.81, 2.1, 136 and 41 in urine. The method developed here does not show interferences or matrix effects produced by endogenous compounds. Micellar media and mobile phases have the advantage of stabilising the compounds, thus preventing photodegradation and allowing the direct injection of biological samples.

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.

Determination of nifedipine and its three principal metabolites in plasma and urine by automated electron-capture capillary gas chromatography.

A sensitive, efficient, linear and reproducible capillary gas chromatographic method with electron-capture detection was developed for the quantitation of nifedipine and its primary metabolite M-I in plasma together with the urinary and principal metabolites M-II and M-III. On-column, rather than split-splitless, injection was employed to obviate oxidative degradation of nifedipine to M-I. The photosensitivity of nifedipine was re-examined under laboratory conditions and nifedipine was found to have a half-life in excess of two days when amber glassware and darkroom manipulations under red light were used. The method can determine nifedipine and its metabolites in plasma and urine after a single oral dose of 5 mg and can be applied to measure M-I production by human liver microsomes.

High performance liquid chromatography analysis of nifedipine and some of its metabolites in hypertensive patients.

Techniques are described for the analysis of nifedipine and three of its metabolites; dehydronifedipine (I), dehydronifedipinic acid (II), and dehydronifedipinolactone (III) in human serum and urine. The analytes are extracted at pH 9 or 3 with ethyl acetate and chromatographed on a C8 reverse-phase column. Recoveries from spiked control serum and urine ranged from 70 to 95% depending on the polarity of the analyte. The coefficient of variation ranged from 10 to 15% (nifedipine, I, and III at 50-200 ng/mL) and 15-20% (II at 200-2000 ng/mL). Accuracy was always within the limits of precision. The metabolism of nifedipine in a group of hypertensive patients receiving long-term nifedipine therapy was compared with that reported for acute administration. Serum nifedipine levels were comparable in both cases, but contrary to previous reports, dehydronifedipine was consistently found in significant amounts. Serum levels of II were considerably elevated but III was not detected either in serum or in urine. Thus, prolonged nifedipine therapy in conjunction with other antihypertensive medications appears to be associated with significant alterations in the metabolism of nifedipine compared with acute administration.

Liquid chromatographic determination of nifedipine in plasma and of its main metabolite in urine.

A high-performance liquid chromatographic method was developed for the assay of nifedipine in plasma and its main metabolite (M-I) in urine. After liquid-liquid extraction nifedipine was chromatographed in a reversed-phase system with ultraviolet detection at 238 nm. The method was sensitive to 2 ng nifedipine per ml plasma and the standard curve was linear to at least 400 ng/ml. Standard deviations did not exceed 8.5%. There was no interference with photodecomposition products or metabolites. M-I was determined in urine after liquid-liquid extraction by ion-pair chromatography with ultraviolet detection at 290 nm. The method was sensitive to 0.02 micrograms M-I per ml urine and the standard curve was linear to at least 5 micrograms/ml. Standard deviations did not exceed 5.0%. The methods were used to study nifedipine disposition in healthy volunteers.

Bimodal column switching liquid chromatographic assay of six metabolites of [14C] felodipine in rat urine.

A liquid chromatographic method using bimodal column switching is presented which allows for the separation of six urinary metabolites of [14C] felodipine and their quantification using an on-line radioactivity detector. Evaluation of the chromatographic conditions was performed with non-labelled reference compounds and UV detection. Pre-separation of the metabolites into two groups, one consisting of carboxylic acid metabolites and the other of the hydroxylated analogues, was performed on underivatized silica. The mobile phase used was optimized with respect to pH and the character of quaternary ammonium ion, and was 0.01 M tetrapropylammonium in 5% (v/v) methanol in 0.05 M phosphate buffer (pH 5.0). Each group was introduced and separated, after band compression, by a gradient of increasing methanol concentration on an octyl-bonded column. The analysis time was 70 min. The method was applied to urine collected from rats (n = 4, 0-24 h) after oral dosing of [14C] felodipine (5 mumol/kg). The urine was analysed with no pre-treatment other than slight dilution. The six metabolites accounted for 58% of the excreted amount (13% of the dose).

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine.

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b] pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane-dichloromethane (7:3) and analysed on a C18 column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 microgram/ml for III and 0.05 microgram/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.

Identification of felodipine metabolites in rat urine.

After intragastric administration of 100 mumol kg-1 [14C]felodipine to rats eight urinary metabolites were isolated. Batch extraction at pH 2.2 and semipreparative reversed-phase liquid chromatography were used for trace enrichment of the metabolites. Trimethylsilylation followed by transesterification with diazomethane blocked the carboxylic acid and alcohol groups selectively before gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. Deuterated derivatives of the metabolites and chemical ionization measurements added complementary structural information. All metabolites reported in this study were formed from oxidized felodipine by ester hydrolysis. Hydroxylation of the pyridine methyl group represented an important metabolic pathway and metabolites oxidized to the corresponding carboxylic acids were detected as well. Lactone formation from hydroxy acid metabolites in urine as a possible analytical artefact is discussed.

Urinary metabolites of felodipine, a new vasodilator drug, in man, dog, rat and mouse.

The urinary excretion of total 14C after oral administration of 25 mg (approximately 1 mumol/kg) 14C-felodipine to man, and intragastric administration (5 mumol/kg) to dog, rat and mouse, was 70, 39, 44 and 53% dose, respectively, in 72 h. Metabolites of felodipine were separated and quantified by h.p.l.c. Unchanged felodipine and its oxidized analogue were not excreted by any of the species studies. Three metabolites, present in all species studied, were isolated from urine and identified as products of the oxidation of felodipine to its pyridine analogue followed by hydrolysis of one or both of the pyridine carboxylic acid esters.

Biotransformation of nifedipine in rat and dog.

Following oral and/or intraduodenal administration, the biotransformation of 14C-labelled nifedipine (dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-pyridine-3, 5-dicarboxylate, Bay a 1040, Adalat, CAS 21829-25-4) has been reinvestigated in rats and dogs (dose: 5 mg/kg body weight in both species) to complete the metabolic data. Thirteen metabolites were isolated from the perfusate and bile of the isolated perfused rat liver model. Their structures were elucidated by spectroscopic methods (FAB-MS, combined GC/MS, NMR). The analyzed samples were used for the chromatographic (HPLC) comparison with urine and bile from the in vivo studies. The metabolites identified in rat urine (oral dose) account for 47.4% of the dose administered. 82.8% (rat) and 62.8% (dog) of the dose, resp., could be attributed to known structures in urine and bile following intraduodenal administration. Based on the structures identified the following biotransformation steps occurred: dehydrogenation of the 1,4-dihydropyridine system, hydroxylation of the methyl groups at 2- or 6-position followed by glucuronidation or by subsequent oxidation to the carboxylic acid, and oxidative ester cleavage.

Nonlinear first-pass metabolism of nifedipine in healthy subjects.

1. A dose-range study was carried out with nifedipine (NF) in 11 healthy subjects, who received orally 5, 10 and 20 mg in capsules (cap) and 20 mg as slow-release tablet. 2. The dose-normalized area under the curve (AUC) of NF showed significant nonlinearity: AUC of 5 mg (cap) was 312 +/- 179, of 10 mg (cap) 357 +/- 186 and of 20 mg (cap) 424 +/- 174 (ng.h/ml). The AUC of the tablet was significantly lower than that of 10 and 20 mg (cap), but not different from 5 mg (cap). 3. The dose-normalized AUC of the pyridine metabolite (M-0) significantly decreased with increasing dose: AUC of 5 mg (cap) was 244 +/- 88, of 10 mg (cap) 194 +/- 96 and of 20 mg (cap) 120 +/- 37 (ng.h/ml). 4. The excretion of M-I (which is ester-hydrolysed M-0) into urine was not different for any of the doses. 5. It is concluded that NF exhibits nonlinear first-pass metabolism, which concomitantly affects the formation of M-0, but not that of M-I.

Metabolism and pharmacokinetics of barnidipine hydrochloride, a calcium channel blocker, in man following oral administration of its sustained release formulation.

1. The metabolism and pharmacokinetics of barnidipine hydrochloride, a 1, 4-dihydropyridine calcium antagonist were evaluated following single oral administration of a sustained release formulation (SR) capsule comprising of quick and slow release pellets to healthy male volunteers. 2. Various metabolites were identified and quantitated by newly established GC-MS analytical methods. Major metabolites were the hydrolyzed product of the benzyl-pyrrolidinyl ester (M-3) in plasma and its oxidized pyridine product (M-4) in plasma and urine. The pyridine form of unchanged barnidipine and the N-debenzylated product were observed as minor metabolites. Therefore, the primary metabolic pathways in man are (a) hydrolysis of the benzylpyrrolidine ester, (b) N-debenzylation, and (c) oxidation of the dihydropyridine ring. 3. When the SR and normal capsules were administered at a dose of 10 mg to six subjects in a crossover design, AUC 0-infinity of unchanged drug, M-3 and 4 in each subject receiving the SR were 97 +/- 15, 85 +/- 31 and 76 +/- 21% respectively of those subjects receiving the normal formulation. The sum of the excretion of urinary metabolites for the SR formulation was 65 +/- 6% of that for the normal formulation. These data suggest that the absorption of the SR formulation is slightly reduced but that its bioavailability is comparable to that of the normal formulation.

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.

Biotransformation of amlodipine. Identification and synthesis of metabolites found in rat, dog and human urine/confirmation of structures by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

Metabolism of the dihydropyridine calcium antagonist (R,S)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbony l-5- methoxycarbonyl- 6 -methyl- 1,4-dihydropyridine (amlodipine) has been studied in animals and man using 14C-labelled drug. The metabolite patterns are complex; 18 metabolites have been isolated from rat, dog and human urine. Based on chromatographic and mass-spectral evidence, structures have been proposed for the main metabolites and confirmed by synthesis of unambiguous reference compounds. Comparison of all reference compounds and isolated metabolites was made by gas chromatography-mass spectrometry pressure liquid chromatography on-line thermospray-mass spectrometry of underivatised compounds directly in urine. The metabolites are largely pyridine derivatives. The methods used in structure designation are presented, along with the proposed route of metabolism, which indicates that the metabolic pattern for amlodipine in man has features in common with those of both rat and dog.

Metabolism of nilvadipine, a new dihydropyridine calcium antagonist, in rats and dogs.

1. The metabolic profiles of nilvadipine in the excreta of male rats and dogs were studied after i.v. and oral dosing. Six types of metabolites were isolated and identified from the urine of male rats and dogs or bile of rats. 2. Several metabolites were detected in the urine (12) and bile (17) of rats by two-dimensional t.l.c., after dosing with 14C-nilvadipine. The metabolic profiles in the excreta of rats and dogs were qualitatively similar but quantitative differences were observed. 3. The main metabolites were products of (i) oxidation of the 1,4-dihydropyridine ring to the corresponding pyridine, (ii) hydrolysis of the 5-isopropyl ester or 3-methyl ester group to carboxylic acid, and/or (iii) hydroxylation of the 6-methyl group or methyl group of the isopropyl ester chain. 4. Minor metabolites were products of hydrolysis from the 5-isopropyl ester to the carboxylic acid having a dihydropyridine ring, or reduction of the 3-nitro group of the phenyl moiety having a pyridine ring.