Reverse Phase HPLC Determination and Murine Pharmacokinetics of Arabinosyl-5-azacytosine

Abstract

A sensitive and specific reverse phase HPLC assay has been developed to measure the new antitumor agent arabinosyl-5-aza-cytosine (ara-AC) in biological fluids at concentrations as low as 50 ng/ml (0.2 μM). This assay also detects arabinosyl-N-formyl-guanylurea (AGU-CHO), the initial hydrolytic metabolite of ara-AC. 2′-Deoxy-5-azacytidine, an analogue with similar chemical stability, is used as an internal standard. Chromatographically interfering plasma ribosides are removed by solid phase extraction on a phenyl boronic acid cartridge. Separation of ara-AC, AGU-CHO and internal standard is then accomplished isocratically (1% CH₃CN in 10 mM pH 6.8 phosphate buffer) on fully carbon loaded and end-capped C₈ and C₁₈ columns connected in tandem. The compounds of interest are detected by UV absorption at 240 nm and total analysis time is 20 min. This assay has been used to determine bolus dose plasma kinetics in male BDF₁ mice given 200 mg/kg ara-AC as a tail vein injection. Plasma elimination of the ara-AC is triphasic with a terminal phase half-life of 52 min and the elimination of the AGU-CHO metabolite parallels that of the parent drug. Analysis of ara-AC in human plasma indicates that this method is suitable for determining drug disposition and pharmacokinetics in human subjects.     

Separation of Xanthine Derivates by High Pressure Liquid Chromatography and Application to Plasma Analysis

Abstract

High pressure liquid chromatography (HPLC) methods were developed for separation and plasma analysis of ten xanthine derivates. Separation was evaluated on silica column and on three different reverse phase columns, with optimum conditions obtained on C₆ spherisorb column using isocratic elution with phosphate buffer 10^?2 M, pH 2.7 - acetonitrile mixture (80/20 V/V). Determination of these xanthine derivates in plasma for therapeutic control was studied.     

The Separation of 12-Molybdophosphoric Acid from 12-Molybdosilicic Acid by Reverse Phase Liquid Chromatography

Abstract

The separation of 12-molybdophosphoric Acid (12-MPA) from 12-molybdosilicic (12-MSA) acid by reverse phase liquid chromatography is described with UV-visible detection. The system utilizes a commercially available styrene-divinylbenzene column (Hamilton PRP-1) and a acetonitrile-water mobile phase which is 0.1 F in HCLO₄ and 1.2 × 10^?2 in molybdate ion. In solutions with acetonitrile concentrations of between 30 and 35% (V/V), 12-MPA is completely retained on the column and 12-MSA has a capacity factor between 1.2 and .6. In solutions with acetonitrile concentrations of between 50 and 60% (V/V) 12-MSA is unretained and 12-MPA has a capacity factor between 2.2 and 1.6.

The detection limit is 5 × 10^?7 M phosphate or silicate anion in 100 uL of injected sample. The linear dynamic range extends to 1.3 × 10^?5 M for either anion. The relative standard deviation of the technique at the 5 × 10^?6 M level is 2% for both silicate and phosphate. The analysis of phosphate in silicate rock is described.     

SYNTHESIS AND CHEMISTRY OF SOME NEW 2-MERCAPTOIMIDAZOLE DERIVATIVES OF POSSIBLE ANTIMICROBIAL ACTIVITY

4,5-Diaryl-2,3-dihydro-2-mercaptoimidazoles (2a–e) were synthesized. They reacted with chloroacetic acid in gl. acetic acid/Ac ₂ O in presence of anhyd. sodium acetate afforded 5,6-diaryl-2,3-dihydro-imidazo[2,1-b]thiazol-3-ones (3a–d). Also these compounds were prepared by the action of chloroacetyl chloride on compounds (2) in pyridine. Compounds (3a–d) on condensation with aromatic aldehydes yield 2-arylmethylene-5,6-diaryl-2,3-dihydroimidazo[2,1-b]-thiazol-3-ones (4a–q). The latter compounds were prepared directly by the reaction of (2) with chloroacetic acid and the aromatic aldehydes. Compounds (3a–d) coupled with aryldiazonium salts in pyridine to give 2-arylhydrazono-5,6-diaryl-2,3-dihydroimidazo[2,1-b]thiazol-3-ones (5a–r). Also compounds (2) when reacted with 2 or 3-bromopropionic acid afford 2,3-di-hydro-5,6-diaryl-2-methylimidazo[2,1-b]thiazol-3-ones (6a–d) and 2,3-di-hydro-6,7-diaryl imidazo-[2,1-b]-1,3-thiazin-4-ones (7a–d), respectively. Compounds (3, 6, and 7) have been cleaved by aromatic amines to give the corresponding 2-(4′,5′-diaryl-2′,3′-dihydroimidazol-2′-yl)thioacetanilide (8a–f), 2-(2′,3′-dihydro-4′,5′-diaryl imidazol-2′-yl)thiopropionamide (9a–c), and 3-(2′,3′-dihydro-4′,5′-diaryl-imidazol-2′-yl)thiopropionamide (10a–d) respectively. All the prepared compounds show considerable antimicrobial activity against bacteria, yeast, and fungi.     

Paired-Ion High Pressure Liquid Chromatography of Methotrexate and Metabolites in Biological Fluids

Abstract

A paired-ion high pressure liquid chromatography procedure (HPLC) is described for the separation of methotrexate and its major metabolites (7-hydroxymethotrexate, 4-amino-4-deoxy-N^lO-methylpteroic acid, methotrexate diglutamate, methotrexate triglutamate), and therapy-related compounds (Diamox and 5-methyltetrahydrofolate). The mobile phase consisted of a 70% solution of 5 mM hexanesulfonic acid, pH 3.75, and 30% methanol eluted on a reverse phase column or columns and monitored at 305 nm or 280 nm UV absorption. The lower limit of sensitivity for methotrexate and 7-hydroxymethotrexate in plasma was 2 ng/ml.     

Electrochemical oxidation of 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-d] pyrimidine-4,6-dione (oxipurinol) at the pyrolytic graphite electrode

The electrochemical oxidation of 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-d] pyrimidine-4,6-dione (oxipurinol) at the pyrolytic graphite electrode (PGE) has been studied. Oxipurinol exhibits up to three voltammetric oxidation peaks at the PGE between pH 1–12. The first pH-dependent peak (peak I_a) is proposed to be an initial, irreversible 2e-2H⁺ reaction to give 5,6-dihydro-4H-pyrazolo[3,4-d] pyrimidine-4,6-dione. This primary product further reacts by two routes. The major route, accounting for ca. 90% of the latter compound, involves a Michael addition of water followed by further electrochemical oxidation and hydrolysis to give 5,6-dihydro-5,6-dihydroxy-5-carboxy-6-diazenouracil. The minor route involves further electrochemical oxidation of 5,6-dihydro-4H-pyrazolo[3,4-d]-pyrimidine-4,6-dione in a 2e-2H⁺ reaction to give 4,5,6,7-tetrahydro-3H-pyrazolo[3,4-d]-pyrimidine-3,4,6-trione.Decomposition and, generally, additional electrochemical reactions of 5,6-dihydro-5,6-dihydroxy-5-carboxy-6-diazenouracil result in the formation of alloxan, parabanic acid, 6-diazo-isobarbituric acid and 5′-hydroxy-5-carboxy-6,6′-azouracil. The two latter compounds have never previously been reported. Decomposition of 4,5,6,7-tetrahydro-3H-pyrazolo[3,4-d]pyrimidine-3,4,6-trione results in formation of uracil-5-carboxylic acid.Detailed reaction schemes have been proposed to explain the observed electrochemistry and the formation of the observed products.     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography.

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6 ,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid-liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate-acetonitrile-methanol-nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C18 reversed-phase column. The standard curves were linear in the range 50-5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2-14%. In the range 50-5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.     

(all-E)-12′-Apozeaxanthinol,Persicaxanthine und Persicachrome

( all-E)-12′-Apozeanthinol, Persicaxanthine, and Persicachromes Reexamination of the so-called ‘persicaxanthins’ and ‘persicachromes’, the fluorescent and polar C₂₅-apocarotenols from the flesh of cling peaches, led to the identification of the following components: (3R)-12′-apo-β-carotene-3,12′-diol ( 3 ), (3S,5R,8R, all-E)- and (3S,5R,8S,all-E)-5,8-epoxy-5,8-dihydro-12′-apo-β-carotene-3,12′-diols (4 and 5, resp.), (3S,5R,6S,all-E)-5,6-epoxy-5,6-dihydro-l2′-apo-β-carotene-3,12′-diol =persicaxanthin; ( 6 ), (3S,5R,6S,9Z,13′Z)-5,6-dihydro-12′apo-β-carotene-3,12′-diol ( 7 ; probable structure), (3S,5R,6S,15Z)-5,6-epoxy-5,6-dihydro-12′-apo-β-carotene-3,12′-diol ( 8 ), and (3S,5R,6S,13Z)-5,6-epoxy-5,6-dihydro-12′-apo-β-carotene-3,12′-diol ( 9 ). The (Z)-isomers 7 – 9 are very labile and, after HPLC separation, isomerized predominantly to the (all-E)-isomer 6 .     

High Performance Liquid Chromatographic Analysis of Rifampin and Related Impurities in Pharmaceutical Formulations

Abstract

High pressure liquid chromatography was employed for the assay of rifampin in capsules. A reverse phase RP-2 column and a mobile phase of 48% methanol, 5% tetrahydrofuran and 47% 0.05 M ammonium formate (pH 7.3) were used with detection at 254 nm. Rifampin was separated from all its major degradation products and quantitated.     

Partial Synthesis and Characterization of the Mono-and Diepoxides of β-Cryptoxanthin

β-Cryptoxanthin ( 1 ) was acetylated and then epoxidized with monoperoxyphthalic acid. After hydrolysis, repeated chromatography, and crystallization, (3S,5R,6S)-5,6-epoxy-β-cryptoxanthin ( 3 ), (3S,5S,6R)-5,6-epoxy-β-cryptoxanthin ( 4 ), (3R,5′R,6′R)-5′,6′-epoxy-β-cryptoxanthin ( 5 ), (3S,5R,6S,5′R,6′S)-5,6:5′,6′-diepoxy-β-cryp-toxanthin ( 6 ), and (3S,5S,6R,5′S,6′R)-5,6:5′,6′-diepoxy-β-cryptoxanthin ( 7 ) were isolated as main products and characterized by their UV/VIS, CD, ¹H- and ¹³C-NMR, and mass spectra. The comparison of the carotenoid isolated from yellow, tomato-shaped paprika (Capsicum annuum var. lycopersiciforme flavum) with 3–5 strongly supports the structure of 3 for the natural product.     

Quantitation of Tamoxifen, 4-Hydroxytamoxifen,and N-Desmethyltamoxifen in Human Plasma by High Performance Liquid Chromatography

Abstract

A reverse phase high performance liquid chromatography assay used for quantitative analysis of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen extracted from human plasma was developed. Plasma samples were spiked with an internal standard, nafoxidine, extracted with 2% butanol in hexane, and activated to fluorescence by exposure to high intensity short-wave ultraviolet (254 nm) light. Aliquots of extracted plasma were then injected onto a C18 reverse phase column and eluted isocratically with a mobile phase of water and triethylamine in methanol. Fluorescence of activated tamoxifen and its metabolites was measured at 266 nm. Peak height ratios were used to quantitate tamoxifen and its metabolites from extracted human plasma specimens.     

Tribute to Prof. Roy Edward Bruns,Universidade Estadual de Campinas

Abstract

Arrhythmic patients treated with quinidine have been found to ingest caffeine from various sources. Caffeine interferes with the quantitation of quinidine in plasma samples. This method for determination of quinidine in plasma is based on high performance isocratic liquid chromatography with the use of a C-18 bonded reverse phase column at room temperature. Unlike some liquid chromatographic procedures for quinidine, caffeine does not interfere. Quinidine is extracted from alkalinized plasma into benzene. The benzene layer is removed and evaporated to dryness under nitrogen at 50 C. The residue is reconstituted with methanol and injected into the column. The mobile phase is 30/70 (v/v) mixture of methanol/0.4% glacial acetic acid. Analysis can be completed within 10 minutes. The procedure is sensitive (0.5mg/L) and is well reproducible (CV= 3.5% for a 2 mg/L concentration in plasma).     

A Reverse Phase HPLC Assay for the Determination of Calcium Pantothenate Utilizing Column Switching

Abstract

A reverse phase HPLC assay utilizing column switching has been developed and validated for the determination of calcium pantothenate (CP) in several multivitamin tablet formulations. The reverse phase system utilizes a DuPont Zorbax C-8 analytical column, an automatically switched and backflushed Brownlee RP-18 guard column for the elimination of a highly retained excipient peak, 88:12 0.25M phosphate buffer:MeOH mobile phase, and 214 nm detection. Sample preparation and the switched column chromatography cycle each require approximately 15 minutes. A spiked recovery study showed linearity over the 50–150% of theory concentration range. Average recovery was 99.7%. Assay precision studies yielded sample RSD's ranging from 0.8 to 2.3%. Results obtained by this method are comparable to those obtained by the USP method.     

Assay of Yohimbine in Human Plasma Using High Performance Liquid Chromatography with Electrochemical Detection

Abstract

Yohimbine is a selective α₂ adrenoreceptor antagonist used in the study of α₂ adrenoreceptors in man. In order to better improve administration regimens for the study of yohimbine in man, we have developed an assay for the determination of yohimbine in plasma utilizing reverse phase high performance liquid chromatography with electrochemical detection. Using a C₁₈ column and a methanol:acetate (60:40) mobile phase, we detected yohimbine in plasma following a simple chloroform extraction. Reserpiline was used as an internal standard. The assay was linear over a concentration range of 50–250 ng/ml in spiked plasma and had a lower limit of sensitivity of 10 ng/ml. It was used to detect yohimbine in plasma sampled from 4 volunteers during an infusion of the alkaloid.     

Liquid Chromatographic Analysis of Pentobarbital

Abstract

A method is described for a one step acetonitrile precipitation of serum or plasma and subsequent analysis of pentobarbital by reverse phase HPLC. The results of using two internal standards, N,N-Diethyl-m-toluamide and 5-(p-Methylphenyl)-5-phenylhydantoin are compared. Internal standard is added to serum (as little as 25 μL) and vortex-mixed with acetonitrile followed by centrifugation. An aliquote of the supernatant is analyzed on a C18 reverse phase column eluted with metanol/0.05 M (NH₄)₂HPO₄, pH 8/water (55/20/25, v/v/v). The effluent is monitored at 220 nm.     

Synthese und Chiralität von (5S, 6R)-5,6-Epoxy-5,6-dihydro-β,β-carotin und (5R, 6R)-5,6-Dihydro-β,β-carotin-5,6-diol,einem Carotinoid mit ungewöhnlichen Eigenschaften

Synthesis and Chirality of (5S,6R)-5,6-Epoxy-5,6-dihydro-β,β-carotene and (5R,6R)-5,6-Dihydro-β,β-carotene-5,6-diol, a Compound with Unexpected Solubility Characteristics Wittig-condensation of azafrinal ( 1e ) with the phosphorane derived from 7 leads to a (1:3)-mixture of (E)-9′- and (Z)-9′-β,β-carotene-diol 3 , from which pure and optically active 3 ((5R,6R)-5,6-dihydro-β,β-carotene-5,6-diol) has been isolated as bright violet leaflets, m.p. 168°. Due to the trans-configuration of the diol moiety and to severe steric hindrance, hydrogen bonding is reduced to such an extent, that 3 behaves much more as a hydrocarbon than as a diol. There is good evidence that the so-called ‘β-oxycarotin’ obtained by Kuhn & Brockmann [15] by chromic acid oxidation of β, β-carotene is the corresponding racemic cis-diol. 3 has been converted into (5S, 6R)-5,6-epoxy-5.6-dihydro-β,β-carotene ( 4 ), m.p. 156°. This transformation establishes for the first time the chirality of a caroteneepoxide (without other O-functions). Full spectral and chiroptical data including a complete assignement of ¹³C-chemical shifts for azafrin methyl ester and 3 are presented.     

Solid-Phase Extraction of Carbamazepine and Two Major Metabolites from Plasma for Analysis by HPLC

Abstract

A rapid, sensitive and simple to operate HPLC method for the simultaneous determination of carbamazepine, carbamazepine 10,11-epoxide and 10,11-dihydro-10,11-trans-dihydroxycarbamazepine in plasma is described. The drug and its metabolites are extracted from plasma using commercially available reversed-phase octadecylsilane bonded-silica columns (Bond Elut C₁₈, 2.8 ml capacity). Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile - methanol - water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Waters Assoc. Nova-Pak C₁₈ column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Waters Assoc. Z-module RCSS and protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak μBondapak C₁₈ insert. Using ultraviolet detection at 214 nm, levels in the region of 50–100 ng/ml for CBZ and its metabolites can be measured with only 250 μl of plasma. The method has been used to determine steady-state concentrations of the drug and its metabolites in paediatric patients.     

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A single LC–MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion‐pair solid‐phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed‐phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile–15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]⁺/60.1 for metformin and m/z 467.1 [M + CH₃COO]^?/329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.     

Absolute Konfiguration von Antheraxanthin, «cis-Antheraxantliirt» und der diastereomeren Mutatoxanthine

Absolute Configuration of Antheraxanthin, ‘cis-Aritheraxanthin’ and of the Stereoisomeric Mutatdxanthins The assignement of structure 2 to antheraxanthin (all-E)-(3 S, 5 R, 6 S, 3′ R)-5,6-epoxy-5,6-dihydro-β,β-carotene-3,3′-diol and of 1 to ‘cis-antheraxanthin’ (9Z)-(3 S, 5 R, 6 S, 3′ R)-5,6-epoxy-5,6-dihydro-β,β-carotene-3,3′-diol is based on chemical correlation with (3 R, 3′ R)-zeaxanthin and extensive ¹H-NMR. measurements at 400 MHz. ‘Semisynthetic antheraxanthin’ ( = ‘antheraxanthin B’) has structure 6 . For the first time the so-called ‘mutatoxanthin’, a known rearrangement product of either 1 or 2 , has been separated into pure and crystalline C(8)-epimers (epimer A of m.p. 213° and epimer B of m.p. 159°). Their structures were assigned by spectroscopical and chiroptical correlations with flavoxanthin and chrysanthemaxanthin. Epimer A is (3 S, 5 R, 8 S, 3′ R)-5,8-epoxy-5,8-dihydro-β,β-carotene-3,3′-diol ( 4 ; = (8 S)mutatoxanthin) and epimer B is (3 S, 5 R, 8 R, 3′ R)-5,8-epoxy-5,8-dihydro-β,β-carotene-3,3′-diol ( 3 ; = (8 R)-mutatoxanthin). The carotenoids 1 – 4 have a widespread occurrence in plants. We also describe their separation by HPLC. techniques. CD. spectra measured at room temperature and at ? 180° are presented for 1 – 4 and 6 . Antheraxanthin ( 2 ) and (9Z)-antheraxanthin ( 1 ) exhibit a typical conservative CD. The CD. Spectra also allow an easy differentiation of 6 from its epimer 2 . The isomeric (9Z)-antheraxanthin ( 1 ) shows the expected inversion of the CD. curve in the UV. range. The CD. spectra of the epimeric mutatoxanthins 3 and 4 (β end group) are dissimilar to those of flavoxanthin/chrysanthemaxanthin (ε end group). They allow an easy differentiation of the C (8)-epimers.     

5,6-二氢-5-氧杂-1,3-二氮杂菲衍生物的合成

以2,3-二氢-3-氧代-1,3-苯并(c)-α-吡喃(1)为起始原料, 在氢化钠作用下, 通过与羰基α-氢的Claisen缩合反应, 得到3-乙酰基-2,3-二氢-3-氧代-1,3-苯并(c)-α-吡喃(2), 所得β-二酮与脲、硫脲和脒衍生物分别进行缩合关环, 生成5,6-二氢-5-氧杂-1,3-二氮杂菲衍生物3和4. 在相同的条件下, 吡喃酮1与草酸二乙酯进行缩合反应, 给出3-乙氧乙二酰 基-2,3-二氢-3-氧代-1,3-苯并(c)-α-吡喃(5), 选择3-氨基吡唑、2-氨基咪唑、3-氨基三唑、2-氨基苯并咪唑和3,5-二氨基吡唑-4-偶氮苯与5缩合, 分别环合成5,6-二氢-5-氧杂-1,3-二氮杂菲并和五元含氮杂环衍生物6～10. 所合成的新化合物均经核磁共振光谱、红外光谱及元素分析证明其结构.