Application of solid-phase extraction to the isolation and determination of paracetamol and its metabolites

A series of clean-up columns for solid-phase extraction (SPE) were packed with various C₁₈ phases of different physico-chemical surface properties. These SPE columns were used for the isolation and determination of paracetamol and its metabolites from biological samples. The packing with a monomeric structure of chemically bonded phase showed the best recovery of tested substances in urine.     

替加氟有关物质的色谱-质谱结构鉴定

目的采用色谱-质谱联用技术鉴定替加氟有关物质,为其工艺和质量控制提供参考依据。方法采用十八烷基硅烷键合硅胶的填充剂,流动相为乙腈-甲醇-10 mmol·L-1醋酸铵缓冲溶液,对替加氟有关物质进行分离;采用电喷雾正离子化-飞行时间质谱法测定各有关物质的准确质量,三重四极杆质谱测定子离子特征,并经解析鉴定各杂质结构。结果与结论替加氟与其有关物质分离良好,检测出4个主要有关物质均为替加氟合成的起始原料,是母核基本未发生变化,仅5位被不同基团取代的衍生物。     

Evaluation of analytical methods: Liquid chromatography of cefalexin

A comparative study of three isocratic liquid Chromatographic methods for the analysis of cefalexin is described. Two methods, taken from the European Pharmacopoeia and the US Pharmacopeia, use classical alkyl bonded phases (C₁₈) as the stationary phase. The third method uses poly(styrene-divinylbenzene). Poor reproducibility of the selectivity towards cefalexin and its related substances was observed for the C₁₈ columns. In both C₁₈ methods, none of the columns was able to separate all the potential related substances from cefalexin under the prescribed LC conditions. The copolymer columns on the other hand showed good selectivity, independent of the origin or age of the columns. Four bulk samples of cefalexin were analysed by these three methods and the results were compared. Although the selectivity of polymer columns was better, for the samples examined no significant differences were observed for the assay results obtained with the three methods.     

Comparative studies on immunologically and non-immunologically produced slow-reacting substances from man, guinea-pig and rat.

1 Slow-reacting substance of anaphylaxis (SRS-A) was produced by antigen challenge of passively sensitized human lung and actively sensitized guinea-pig lung. 2 A slow-reacting substance (SRS) was prepared from the peritoneal fluid of rats treated with calcium ionophore A23187. 3 These substances were extensively purified by charcoal adsorption, Sephadex G-15 gel filtration, ether extraction and reverse phase high pressure liquid chromatography. 4 The three substances are pharmacologically, chemically and chromatographically indistinguishable. 5 Our data suggest that the same SRS entities are released from a variety of tissues and that these acidic lipids may have a wider physiological significance than just anaphylaxis.     

Normal phase HPLC as a model system of specific benzodiazepine-receptor binding

The hypothesis that benzodiazepines interact with their receptors in the CNS via hydrogen bonding mediated by the carbonyl and imine groups has been studied by correlating a physicochemical parameter affected by hydrogen bonds potentially accepted or donated by benzodiazepines with their binding constants to synaptosomal rat brain membranes. The reference physicochemical system chosen was normal phase high-performance liquid chromatography on mu-Bondapack NH2 (propylamine groups chemically bonded to porous silica) eluted with mixtures of n-hexane and 2-propanol. A strong correlation has been found for binding of the implicated groups to the column and to the receptor.     

On-line HPLC method for clean-up and analysis of hydrocortisone and sulconazole nitrate in a cream

The application of a new on-line high-performance liquid Chromatographic method for the clean-up and analysis of drugs in semi-solid formulations was investigated. A short column of a chemically bonded stationary phase (10 μm octadecylsilane) mounted into the sample loop position of the high pressure injection valve was used as the primary clean-up step. The elution behavior of commonly used cream excipients on the loop column as a function of various combinations of water, methanol and THF was studied using a refractive index detector. Typically, samples of the cream were dissolved in methanol-THF and injected onto the loop column. The drug substances were eluted onto the analytical column with predetermined volumes of the mobile phase. The relatively non-polar cream excipients were retained on the loop column. At this time the injection valve was switched, isolating the loop column and the retained excipients were eluted off to waste with a strong solvent (methanol-THF). Loss of efficiency due to the insertion of the loop column was negligible. This technique was applied to the simultaneous analysis of a corticosteroid (hydrocortisone) and a hydrophobic amine (sulconazole nitrate) in a topical cream formulation.     

HPLC法测定索法酮的有关物质

目的建立索法酮有关物质的测定方法。方法采用高效液相色谱法,以十八烷基硅烷键合硅胶为固定相,用三乙胺调节流动相pH值至3.5,检测波长为348 nm。结果线性范围为1.23～3.28μg.mL-1,方法专属性强、灵敏度高、精密度好。结论该方法适用于索法酮有关物质的测定。     

HPLC法测定曲安奈德益康唑乳膏中的有关物质

目的:建立HPLC法测定曲安奈德益康唑乳膏中有关物质的方法。方法:采用高效液相色谱法,色谱条件为:色谱柱以十八烷基硅烷键合硅胶为填充剂(Kromasil100-5C18,4.6mm×250mm,5μm),柱温为45℃;以0.077%醋酸铵缓冲液(用磷酸调节pH值至3.0)-甲醇(80∶20)为流动相A;甲醇-乙腈(40∶60)为流动相B;流速为1.0mL/min;梯度洗脱;检测波长为240nm和225nm。结果:在225nm检测波长下,阴性样品色谱峰、曲安奈德峰及其杂质峰对硝酸益康唑及其杂质检测无干扰,加已知杂质的供试品溶液中益康唑、益康唑杂质A、杂质B、杂质C、咪康唑及未知杂质峰和相邻色谱峰分离度均符合要求;在240nm检测波长下,阴性样品色谱峰、硝酸益康唑及其杂质色谱峰对曲安奈德及其杂质检测无干扰,加已知杂质的供试品溶液中,曲安奈德、曲安奈德杂质A(曲安西龙)、杂质B、杂质C及未知杂质与相邻色谱峰分离度均符合要求。结论:经过对拟定的有关物质方法的方法学验证,表明本方法具有重现性和科学性,可用于曲安奈德益康唑乳膏有关物质检查。     

Analysis of designer drugs detected in the products purchased in fiscal year 2006

Many psychotropic substances are easily available in Japan via the Internet, thus the spread of drug abuse is becoming more serious problem. To avoid drug abuse, 32 substances have been controlled in Japan since April in 2007 by the Pharmaceutical Affairs Law as designated substances (Shitei-Yakubutsu, classified as 11 tryptamines, 11 phenethylamines, 2 piperazines, 6 alkyl nitrites, 1 diterpene and 1 plant). Although the distributions of these drugs have been decreased through this regulation, new designer drugs are still being found. In this study, we detected 7 designer drugs in 15 products, which purchased just before the amendment of the law, by NMR, GC-MS and LC-MS analyses. Three methylone derivertives (1-(3,4-methylenedioxyphenyl-2-(pyrrolidin-1-yl)-1-pentanone: MDPV, 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one: bk-MBDB, 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one): bk-MDEA, a MDMA derivative (N-hydroxy-1-(3,4-methylenedioxyphenyl)-2-aminopropane: N-OH MDMA), a methamphetamine derivative (N-methyl-1-(4-fluorophenyl)propan-2-amine: N-Me-4-FMP), a tryptamine derivative (5-methoxy-N-ethyl-N-isopropyltryptamine: 5-MeO-EIPT) and indan-2-amine were detected. 5-MeO-EIPT was newly identified in this study.     

HPLC法测定不同产地木香中木香烃内酯含量

目的：建立木香中木香烃内酯含量测定的HPLC法。方法以十八烷基键合硅胶为填充剂,流动相为甲醇—乙腈—水（65∶5∶30）,检测波长225 nm,流速1．0 mL·min－1。结果木香烃内酯在12～120 mg·L－1范围内线性关系良好,回归方程为Y＝2．16×104X＋92064．7（r＝0．9995）,平均加样回收率为98．90％,RSD为1．53％。结论 HPLC法简单易行、准确度高,可用于木香的质量控制,不同产地的木香中木香烃内酯含量差别较大。     

HPLC法测定葡甲胺中的有关物质

目的:采用高效液相色谱方法测定葡甲胺中有关物质。方法:采用强酸性阳离子交换基团键合全多孔不规则形硅胶固定相色谱柱(SCX),流动相为三氟乙酸-甲酸-水(0.05∶0.3∶100),流速0.6 mL.min-1,示差折光检测器,柱温35℃;并采用液相质谱方法对该系统进行分离度检测。结果:使用该系统能够使葡甲胺主峰与有关物质完全分离,分离度均大于2.0;最低检出限为10μg.mL-1。结论:本方法简便、准确、稳定,专属性强,能够对葡甲胺中的有关物质进行有效检测。     

人血浆中乙胺碘呋酮的高效液相色谱法测定

应用改性甲醇(500ml甲醇中加入0.25 ml丁胺,混匀,继加1.0 ml 1:1冰醋酸为流动相,在YWG-CN(腈基键合相)柱上,以回苏灵(dimefline)为内标,对乙胺碘呋酮建立了适合于临床药理和临床化学应用的高效液相色谱测定法。本法简便、快速、灵敏、重现性好,流动相无需脱气并可循环使用。样品用乙醚提取,乙胺碘呋酮及回苏灵的平均回收率均大于97%。测定结果的变异系数,日内小于3.5%,日问小于5.0%。     

Synthesis and anticonvulsant activity of O-alkylated derivatives of 1-(2-naphthyl)-2-(imidazol-1-yl)ethanone oxime

In this study, O-alkylated derivatives of nafimidone oxime chemically, 1-(2-naphthyl)-2-(imidazol-1-yl)ethanone oxime have been synthesized as potential anticonvulsant compounds. O-alkylation of the oxime using hydrochlorides of various dialkylaminoethyl chlorides, methyl chloroacetate and alkyl dihalides gave the O-alkylated derivatives. Anticonvulsant activity of the compounds was determined against pentylenetetrazole induced convulsions in mice. The newly synthesized compounds exhibited moderate to significant activity compared to diazepam.     

Evaluation of the Functional Equivalence of Crospovidone NF from Different Sources. I. Physical Characterization

In the past two decades, three categories of newer disintegrants have come into widespread use. These substances are a synthetic polymer (crospovidone), chemically modified starch (sodium starch glycolate) and cellulose (croscarmellose sodium). Multiple suppliers are now available for each category. Current NF monographs do not provide tests which reflect on their functionality and one cannot assume reliable performance of disintegrants from different sources meeting NF standards. The objective of this study was to identify differences in physical properties thought to be related to functionality among crospovidones from multiple sources. Physical properties examined included particle size and distribution, surface area, porosity and surface morphology. Disintegration and dissolution were performed on a model tablet formulation using either an insoluble or a soluble filler. Substantial differences in particle size and distribution, surface area, porosity and surface morphology were observed which correlated with differences in disintegration time and dissolution rate of the model drug from an insoluble tablet core. None of the differences in physical properties resulted in any differences in the disintegration or dissolution of the model drug from a soluble tablet core.     

Evaluation of the functional equivalence of crospovidone NF from different sources. I. Physical characterization

In the past two decades, three categories of newer disintegrants have come into widespread use. These substances are a synthetic polymer (crospovidone), chemically modified starch (sodium starch glycolate) and cellulose (croscarmellose sodium). Multiple suppliers are now available for each category. Current NF monographs do not provide tests which reflect on their functionality and one cannot assume reliable performance of disintegrants from different sources meeting NF standards. The objective of this study was to identify differences in physical properties thought to be related to functionality among crospovidones from multiple sources. Physical properties examined included particle size and distribution, surface area, porosity and surface morphology. Disintegration and dissolution were performed on a model tablet formulation using either an insoluble or a soluble filler. Substantial differences in particle size and distribution, surface area, porosity and surface morphology were observed which correlated with differences in disintegration time and dissolution rate of the model drug from an insoluble tablet core. None of the differences in physical properties resulted in any differences in the disintegration or dissolution of the model drug from a soluble tablet core.     

Analytical data of designated substances (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan, part I: GC-MS and LC-MS

In the last 10 years, many analogs of narcotic substances have been widely distributed in Japan as easily available psychotropic substances and this has become a serious problem. They have been sold as video cleaners, incense and reagents via the Internet or in video shops. They are not controlled under the Narcotics and Psychotropics Control Law because their pharmacological effects have not yet been proved scientifically. As a countermeasure to prevent the abuse of these substances, the Ministry of Health, Labor and Welfare amended the Pharmaceutical Affairs Law in 2006 so that 31 non-controlled psychotropic substances (11 tryptamines, 11 phenethylamines, 6 alkyl nitrites, 2 piperazines and salvinorin A) and 1 plant (Salvia divinorum) are now controlled as "Designated Substances (Shitei-Yakubutsu)" as of April 2007. Five other compounds (4 phenethylamines and 1 piperazine) were also added to this category in January 2008. In this study, we developed simultaneous analytical methods for these designated substances using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) and present retention times, UV spectra, electron ionization (EI), GC-MS, and electrospray ionization (ESI) LC-MS data.     

Synthesis and pharmacological activity of 3-carboxypicolinic acid amides

A series of substituted amides of 3-carboxypicolinic acid have been synthesized via the interaction of quinolinic acid anhydride with alkyl(aryl)amines. Their structures were studied by ¹H and ¹³C NMR spectroscopy. The direction of the reaction was explained by quantum-chemistry methods. Three compounds demonstrated hemostatic activity. Two substances exhibited anticoagulant properties. Most substances also exhibited weak bacteriostatic activity.     

Design and synthesis of 4H-3,1-benzoxazin-4-ones as potent alternate substrate inhibitors of human leukocyte elastase

4H-3,1-Benzoxazin-4-ones are alternate substrate inhibitors of the serine proteinase human leukocyte elastase (HL elastase) and form acyl enzyme intermediates during enzyme catalysis. We have synthesized a large variety of benzoxazinones using specific methods that have been adapted to achieve the pattern of ring substitution dictated by theoretical considerations. The results of the inhibition of HL elastase by 175 benzoxazinones are reported herein with reference to hydrophobicity constants D, alkaline hydrolysis rates kOH-, inhibition constants Ki, and their component acylation and deacylation rate constants, kon and koff, respectively. The ranges for the compounds are considerable; alkaline hydrolysis rates and kon span 6, koff covers 5, and ki spans 8 orders of magnitude. Multiple regression on this large data set has been used to isolate the contributions of electronic and steric effects, as well as other factors specific to compound stability and elastase inhibition. Essentially, a simple electronic parameter is sufficient to account for almost all the variance in the alkaline hydrolysis data, indicating that electronic factors are the major determinants of this type of benzoxazinone reactivity. Factors that significantly enhance the potency of benzoxazinones I are R5 alkyl groups and electron withdrawal by R2. Bulk in R7 and R8 and compound hydrophobicity are not significant, but substitution in R6 is highly unfavorable as are substituents linked via carbon to C2. The physiochemical factors that underlie these trends in Ki are further analyzed in terms of equations that describe kon and koff. A conclusion that emerges is that chemically stable, potent benzoxazinone inhibitors of HL elastase with inhibition constants in the nanomolar range can be designed with (1) R5 alkyl groups to inhibit enzyme-catalyzed deacylation, (2) small alkyl substituents linked via heteroatoms to C2 to enhance acylation and limit deacylation rates, and (3) strongly electron-donating groups at C7 to stabilize the oxazinone ring to nucleophilic attack. Thus, 2-(isopropylamino)-5-n-propyl-7-(dimethylamino)benzoxazinone 95 has kOH = 0.01 M-1 s-1, which extrapolates to a half-life at pH 7.4 of over 8.5 years, and 2-ethoxy-5-ethylbenzoxazinone 38 has Ki = 42 pM.     

Facile synthesis of platelet-activating factor and racemic analogues containing unsaturation in the sn-1-alkyl chain

Platelet-activating factor, 1 (PAF, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), and octadecyl-PAF were synthesized chemically as the racemates. The sn-1-O-alkyl isomers were isolated after treatment of the racemates with phospholipase A2 and subsequent reacetylation of the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholines released. Analogues of PAF containing unsaturated alkyl moieties at the sn-1 position (2, 4, 5) were synthesized by utilizing the methoxyethoxymethyl protecting group as a novel method for preparing unsaturated alkyl lipids. This procedure provides a facile means for preparing unsaturated either phospholipids of defined structure that may be tritiated to high radiospecific activity for metabolic studies. Unsaturation in the alkyl chain had minimal effect on the bioactivities examined in this study.     

Different in vitro and in vivo tools for elucidating the human metabolism of alpha‐cathinone‐derived drugs of abuse

In vitro and in vivo experiments are widely used for studying the metabolism of new psychoactive substances (NPS). The availability of such data is required for toxicological risk assessments and development of urine screening approaches. This study investigated the in vitro metabolism of the 5 pyrrolidinophenone‐derived NPS alpha‐pyrrolidinobutyrophenone (alpha‐PBP), alpha‐pyrrolidinopentiothiophenone (alpha‐PVT), alpha‐pyrrolidinohexanophenone (alpha‐PHP), alpha‐pyrrolidinoenanthophenone (alpha‐PEP, PV8), and alpha‐pyrrolidinooctanophenone (alpha‐POP, PV9). First, they were incubated with pooled human liver microsomes (pHLM) or pooled human liver S9 fraction (pS9) for identification of the main phase I and II metabolites. All substances formed hydroxy metabolites and lactams. Longer alkyl chains resulted in keto group and carboxylic acid formation. Comparing these results with published data obtained using pHLM, primary human hepatocytes (PHH), and authentic human urine samples, PHH provided the most extensive metabolism. Second, enzyme kinetic studies showed that the initial metabolic steps were formed by cytochrome P450 isoforms (CYP) CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 resulting in pyrrolidine, thiophene or alkyl hydroxy metabolites depending on the length of the alkyl chain. The kinetic parameters indicated an increasing affinity of the CYP enzymes with increase of the length of the alkyl chain. These parameters were then used to calculate the contribution of a single CYP enzyme to the in vivo hepatic clearance. CYP2C19 and CYP2D6 were mainly involved in the case of alpha‐PBP and CYP1A2, CYP2C9 and CYP2C19 in the case of alpha‐PVT, alpha‐PHP, alpha‐PEP, and alpha‐POP.