Determination of debrisoquine and metabolites in human urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric analysis has been developed for the determination of debrisoquine and its metabolites in the urine of healthy individuals (controls) and patients with chronic renal failure. The sensitive and specific assay comprises selected-ion monitoring of the drug and the metabolites 4-hydroxydebrisoquine and 8-hydroxydebrisoquine using guanoxan as the internal standard. The limit of detection is ca. 0.2 microgram/ml. The clinical study shows that the healthy individuals and patients with chronic renal failure can be divided in two groups of extensive metabolizers and poor metabolizers, respectively. The extensive metabolizers excreted large amounts of 4-hydroxydebrisoquine and minor amounts of 8-hydroxydebrisoquine. The poor metabolizers excreted small amounts of 4-hydroxy metabolite, and no 8-hydroxydebrisoquine was detected in the urine.     

Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies

A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4-hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), debrisoquine/5-hydroxydebrisoquine (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid-phase extraction. The chromatography was performed using a C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05-5 microg/mL for caffeine and paraxanthine, 0.02-2 microg/mL for tolbutamide, 0.1-20 microg/mL for 4-hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5-hydroxydebrisoquine, 5-2500 ng/mL for omeprazole and 5-hydroxyomeprazole, and 1-100 ng/mL for midazolam and 1'-hydroxymidazolam. The intra- and inter-day precision were 0.3-13.7% and 1.9-14.3%, respectively, and the accuracy ranged from 93.5-107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five-probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects.     

Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables

A liquid chromatographic method has been described for the determination of total vitamin C, ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fruits and vegetables. The complete separation of AA and DHAA could be achieved on a C18 column using 0.2 M KH2PO4 (pH adjusted to 2.4 with H3PO4) as the mobile phase at a flow-rate of 0.5 ml/min. Since the detection sensitivity was poor for DHAA even at 210 nm, it was estimated as the difference between the total AA after DHAA reduction and AA content of the original sample, using dithiothreitol (DTT) as the precolumn reductant. The reaction times for the complete conversion of DHAA to AA at room temperatures were 150, 120, 90 and 75 min for 1, 2, 4 and 8 mmol DTT per mmol of DHAA, respectively. The percentage recovery ranged from 81.7 to 105.9. AA contents of some selected fruits and vegetables were analyzed comparatively by liquid chromatography and enzymatic assay to validate the method.     

Liquid chromatographic assay for the simultaneous determination of pyrazinamide and rifampicin in serum samples from patients with tuberculous meningitis

A simple high-performance liquid chromatographic assay for the simultaneous determination of pyrazinamide and rifampicin in serum from patients with tuberculous meningitis is presented. The drugs and internal standard, p-acetamidobenzoic acid, were extracted from the acidified sample containing 2% ascorbic acid at pH 4.2 into dichloromethane-diethyl ether (2:3). The solvent extract was evaporated to dryness with the aid of nitrogen and the residue redissolved in methanol (75 microliters). The concentrate was analysed by a liquid chromatograph using a reversed-phase 30-microns C8 pre-column linked to a 5-microns C8 analytical column with a gradient solvent programme, which delivered 6% to 48% (v/v) acetonitrile in phosphate buffer (10 mM potassium dihydrogenphosphate, pH 3.5) in 10 min at 1.5 ml/min. The eluate was detected at 215 nm. Twelve patients with tuberculous meningitis were given daily chemotherapy, and their serum samples were assayed for pyrazinamide and rifampicin.     

High-performance liquid chromatographic analysis of amiloride in plasma and urine

A high-performance liquid chromatographic method has been developed for amiloride in rabbit plasma and urine which uses a reversed-phase C18 column, a mobile phase (flow-rate 2 ml/min) consisting of 32% acetonitrile in 0.15 M perchloric acid, pH 2.2, and spectrofluorometric detection via excitation at 286 nm. A simple extraction step with ethyl acetate eliminates interfering peaks. Short retention times of about 2.3 and 3.8 min are observed for amiloride and the internal standard, triamterene, respectively. The method can measure 4 ng/ml amiloride in plasma. This assay has been used to explore the pharmacokinetics of amiloride in rabbits. The plasma disposition profile is biexponential after a 50-mg intravenous bolus dose and there is no evidence for saturable elimination at zero-order infusion rates of 1.8, 3.6 and 7.2 mg/h.     

High-performance liquid chromatographic determination of amphotericin B in a liposomal pharmaceutical product and validation of the assay

A validated high-performance liquid chromatographic method is presented to quantitate amphotericin B (AB) in a liposomal pharmaceutical formulation. The analysis is based on the chromatographic separation of AB and 1-amino-4-nitronaphthalene (the internal standard) on a C18 muBondapac reversed-phase column with a mobile phase consisting of a mixture of acetonitrile and 0.02 M ethylenediamine tetra-acetic acid disodium salt at pH 5.0 (45:55, v/v). The chromatographic analysis time is less than 10 min, and the validation of the assay shows that it is selective, accurate, and linear for the concentration range of 2.50 to 7.50 microg/mL with a detection limit of 0.00500 microg/mL. The within-day and between-day relative standard deviation values are 1.26% (n = 18) and 1.25% (n = 8), respectively. The method described conforms to the validation of compendial methods used for finished pharmaceutical products in general and offers a reliable, quick, and cost-effective procedure for examining the consistency or quality-control analysis of AB in liposomal products. It can also be applied for the determination of AB in other nonliposomal lipid-based drug delivery systems that are on the market.     

Selective liquid chromatographic method for determination of fluoxetine in plasma.

A selective and sensitive liquid chromatographic method was developed for the determination of fluoxetine (FLU) in plasma. FLU was isolated from plasma by liquid-liquid extraction. The chromatographic separation was performed on an analytical 250 x 3.9 mm id Novapak C18 column (4 microm particle size) with an isocratic mobile phase consisting of phosphate buffer-acetonitrile-methanol -triethylamine (58 + 30 + 10 + 2, v/v) adjusted to pH 7. Using UV detection at 226 nm, the detection limit for FLU in plasma was 3 ng/mL. No interferences were found with tricyclic antidepressant drugs, which allows this method to be used in clinical studies. The calibration curve was linear over the concentration range of 10-200 ng/mL. The average recovery was about 80% for plasma. The inter- and intraday assay coefficients of variation were <8%.     

Comparison of Chirasil-DEX CB as gas chromatographic and ULMO as liquid chromatographic chiral stationary phase for enantioseparation of aryl- and heteroarylcarbinols

For a broad spectrum of simple chiral alcohols, incorporating a (substituted) (het)aryl building block, enantiomer separation characteristics are reported for both gas chromatography on a Chirasil-DEX phase, and liquid chromatography on an (S,S)-ULMO phase. On this chiral Pirkle-type phase, homochiral enantiomers (mostly R) are eluted first without exception. The elution order R before S appears conserved as a rule also for gas chromatographic separations on Chirasil-DEX, though with some remarkable exceptions indicating a change in the dominant discriminative mechanism. This was shown in the homologous series 1-phenylethanol to 1-phenylhexanol having the point of reversal at C4, while the o-methoxy analogues elute from C1 to C4 already in the reversed order.     

Determination of furosemide in urine samples by direct injection in a micellar liquid chromatographic system

A sensitive, selective and efficient micellar liquid chromatographic (MLC) procedure was developed for the determination of furosemide (4-chloro-N-furfuryl-5-sulfamoylanthranilic acid) in urine samples by direct injection and UV detection. The procedure makes use of a C18 reversed-phase column and a micellar mobile phase of 0.05 mol l(-1) sodium dodecyl sulfate-6% v/v propanol and phosphate buffer at pH 3 to resolve furosemide from its photochemical degradation products. The importance of protecting the standards and urine samples to be analysed from light in the assay of furosemide, avoiding its degradation, was verified. The limit of quantification was 0.15 microg ml(-1) and the relative standard deviation of the inter-day assay was 0.8-0.04% in the 6-82 microg ml(-1) range. Detection of urinary excretion of furosemide was followed up to 12 h after ingestion of the drug by a healthy volunteer. No potential interference from the major metabolite (furosemide acylglucuronide) and its hydrolytic product (4-chloro-5-sulfamoylanthranilic acid) was observed. Commonly administered drugs also did not interfere. The proposed MLC procedure permits the rapid and reproducible measurement of low levels of furosemide in a small amount of urine.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

A high-performance liquid chromatographic assay of the electro-oxidation of purines : Uric Acid and the Nucleotide Drug Tubercidin-5′-Monophosphate

A high-performance liquid chromatographic assay is described for establishing the completeness of electro-oxidation of a purine nucleotide drug tubercidin-5′-monophosphate (TMP) and the sequence of product formation. The assay shows that TMP was completely oxidized after ca. 2 h of electrolysis and that the product distribution continued to change after this time. The same assay was also used to show the sequence of product formation in the electro-oxidation of uric acid. Both separations were isocratic, with a 0.02 M potassium dihydrogenphosphate (pH 4.6–5.1) mobile phase.     

Effective high-performance liquid chromatographic determination of glafenine in plasma: pharmacokinetic application

A high-performance liquid chromatographic method for an effective determination of glafenine and its main metabolite, glafenic acid, is described. The assay involves separate extraction procedures for glafenine and for its metabolite, but the same internal standard (floctafenine) and the same chromatographic conditions (including a 5-micron C8 column, a quaternary solvent mixture of water-acetonitrile-diethylamine-acetic acid and an ultraviolet detector set at 360 nm). For 1 ml of plasma, the detection limit is 0.05 mg/l for glafenine and 0.25 mg/l for glafenic acid. Compared with previously described techniques, this assay uses a very low glafenine linearity range, which allows the true pharmacokinetics of this drug to be described for the first time.     

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.     

Improvement of the chromatographic separation of several 1,4-dihydropyridines calcium channel antagonist drugs by experimental design

A high-performance liquid chromatographic method with diode array detection has been developed and optimized for the separation of five calcium channel blockers belonging to the 1,4-dihydropyridine subgroup (nifedipine and related drugs). The possibility of the simultaneous drug analysis allows a decrease of time during the assay as well as a saving of reagents and solvents. In this work, the effect of four experimental parameters (organic modifier percentage, pH value, concentration of the buffer in the mobile phase, and column temperature) on the chromatographic resolution are investigated by experimental design in order to optimize the chromatographic separation of five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine, and lercanidipine). Fractional factorial design, central composite design, and finally the Multisimplex program are used to establish the optimal conditions in terms of resolution and minimum analysis time. Optimal separation of the five compounds under study is achieved in less than 12 min using a Sulpecosil LC-ABZ+Plus C18 column, a composition of mobile phase of acetonitrile-10mM acetic acid acetate buffer pH 5 (72:28, v/v) at a flow rate of 1 mL/min, a column temperature of 30 degrees C +/- 0.1 degrees C, and a detection wavelength of 238 nm.     

Biological assay and liquid chromatographic method for analysis of moxifloxacin in tablets

A microbiological assay and a liquid chromatographic method were validated for quantitation of moxifloxacin in tablets. The microbiological method consisted of a cylinder-plate agar diffusion assay using Micrococcus luteus ATCC 9341 as the test microorganism and phosphate buffer (0.1M, pH 8.0) as the diluent solution. The response graphs for standard and sample solutions were linear (r = 0.9479), and no parallelism deviations were detected in the tested levels of concentration (4.0, 8.0, and 16.0 microg/mL). The interday precision was 2.73%. Recovery values were between 96.25 and 100.5%. The chromatographic analyses were performed using a Shim-pack CLC-ODS column (250 x 4.6 mm, 5 microm) with a mobile phase consisting of (A) a mixture of phosphoric acid (0.17%, v/v) with tetramethylammonium hydroxide (0.05M) and acetonitrile (95 + 5, v/v) and (B) methanol (55 + 45, v/v) adjusted to pH 3.0. The flow rate was 1.0 mL/min, and detection was made at 294 nm. The method was linear in a range from 12.0 to 42 microg/mL (r = 0.9999), and the interday precision was 1.39%. Recovery ranged between 101.9 and 103.81%. Both validated methods were used to quantify the moxifloxacin content in tablets exposed to ultraviolet radiation, and similar results were obtained.     

Combined liquid and gas chromatographic characterisation of polyglycerol fatty acid esters

In the present study a combined liquid and gas chromatographic technique is described for the analysis of polyglycerol fatty acid esters. Liquid chromatographic fractionation of samples resulted in pure standards of monoesters of di- and triglycerols and diesters of di- and triglycerols. Confirmation of their identity was achieved by LC-MS analysis. Moreover, a chromatographic identification of the mono- and diesters of cyclic diglycerol was proposed. From the isolation of pure esters and their gas chromatographic analysis, it was revealed that co-elution of several compounds occurred. Thus it was shown that prefractionation of the sample using a simplified liquid chromatographic separation, was necessary in order to characterise the esters correctly. In combination with some other chemical analyses, a complete profile of the chemical composition of polyglycerol fatty acid esters can be obtained.     

Versatile isocratic high-performance liquid chromatographic assay for propranolol and its basic, neutral and acidic metabolites in biological fluids

A comprehensive high-performance liquid chromatographic method was developed for quantitating propranolol and its known metabolites in serum, bile and urine. Analysis was performed before and after incubation of the samples with beta-glucuronidase-arylsulfatase to quantitate both free and conjugate forms of the oxidative metabolites. Fractionation of the basic, neutral and acidic metabolites was achieved by differential pH solvent extraction. The basic and neutral metabolites were extracted from the biological samples at pH 10.5 with 2% n-butanol in dichloromethane. Additional clean-up of the basic fraction by back-extraction into dilute acid was needed for those samples that were subjected to enzymatic hydrolysis. The original aqueous sample was titrated with acid to pH 1, followed by extraction of the remaining acidic metabolites into either n-butanol-dichloromethane (with unhydrolyzed serum) or carbon tetrachloride (with all other samples). Chromatographic separation of the metabolites in the different extracts was achieved on a reversed-phase C18 column, using a single isocratic mobile phase consisting of 0.044 M pH 2.7 phosphate buffer, tetrahydrofuran, methanol and acetonitrile, with the addition of n-butylamine as a competing base to control retention volume and peak shape. Detection and quantitation of propranolol and its metabolites in the low nanogram to sub-nanogram range was afforded by fluorescence at a low UV excitation wavelength. The coefficients of variation for replicate assay of spiked samples were uniformly less than 6% for all the analytes.     

Development and substantiation of a liquid chromatographic method for monitoring organic reactions involved in synthesis of 4-methoxyphenylacetic acid

A simple and rapid reversed-phase high-performance liquid chromatographic method for monitoring the reactions involved in two different processes for the production of 4-methoxyphenylacetic acid (PMPA) was developed. Impurity profiles of PMPA were used for fingerprinting of the two different synthetic processes by HPLC. Impurities were separated and determined on a Hypersil C18 column with acetonitrile-0.1 M potassium dihydrogen orthophosphate-triethylamine (40:59.95:0.05, v/v) (pH 3.0) as the mobile phase and detection at 280 nm at ambient temperature. The method was substantiated with respect to accuracy, precision, linearity, robustness, limit of detection and quantification. The method was found to be suitable not only for monitoring the reactions but also for quality assurance of PMPA as it could detect impurities at the level of 4 x 10(-9) g.     

High-performance liquid chromatographic analysis of a novel carbapenem antibiotic in human plasma and urine

High-performance liquid chromatographic methods for quantification of a novel carbapenem anti-infective agent, I, in plasma and urine have been developed, validated, and applied to clinical samples. The carbapenem is stabilized in the matrix by the addition of a non-nucleophilic buffer, rapid freezing, and storage at -70 degrees C. After addition of another carbapenem, II, as internal standard, plasma proteins are precipitated with acetonitrile, which is subsequently extracted from the sample with methylene chloride. A portion of the aqueous phase is injected onto a reversed-phase phenyl column that is eluted with 4% (v/v) acetonitrile in 15 mM ammonium phosphate (pH 7.4). The urine assay entails addition of the internal standard II to buffered urine, which is subsequently extracted with methylene chloride prior to injection of the aqueous phase onto a cation-exchange column. The urine assay mobile phase is 5% v/v tetrahydrofuran in 100 mM sodium acetate (pH 5.4). The detector response at 313 nm is a linear (r greater than 0.99) function of concentration over the ranges 0.50-100 micrograms/ml and 2.0-200 micrograms/ml for the plasma and urine assays, respectively. Thermal degradation products do not interfere with either assay. These assays have proven to be accurate, precise, reproducible, and rugged during clinical sample analyses.     

Pre-column derivatization of sulfa drugs with fluorescamine and high-performance liquid chromatographic determination at their residual levels in meat and meat products.

A rapid, sensitive and selective high-performance liquid chromatographic method is described for simultaneous determination of eight sulfa drugs in meat and meat products using pre-column derivatization with fluorescamine. The drugs are sulfisomidine, sulfadiazine, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. The method includes blender extraction of 3-g samples with chloroform, partition with 3 M hydrochloric acid, derivatization with fluorescamine at pH 3.0 and subsequent high-performance liquid chromatographic analysis on a C18 column with fluorescence detection at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. The drugs were separated with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C. The average recovery from samples fortified at 0.1 ng/g was 92.6% with a coefficient of variation of 6.2%. The detection limit was 0.01 ng/g for sulfaquinoxaline and 0.005 ng/g for the other seven drugs. The method was field-tested in a survey of 37 samples including beef (five), pork (seven), chicken (seven), ham (five), sausage (eight), bacon (two) and roast beef (three). Sulfadimidine was detected in one pork sample at the level of 0.295 ng/g and in ham at 0.178 ng/g.