Isolation and identification of 8-hydroxypraziquantel as a metabolite of the antischistosomal drug praziquantel

Praziquantel, a broad spectrum anthelmintic drug, is extensively metabolized in the liver, yielding mainly monohydroxylated and dihydroxylated phase-I metabolites. However, the exact chemical structure of most metabolites is still unknown. One of these unidentified phase-I metabolites was isolated from human urine by high performance liquid chromatography using an isocratic separation method. This metabolite was identified as 8-hydroxypraziquantel. For the structure elucidation, electrospray ionization–mass spectrometry, ¹H and ¹³C NMR spectroscopy have been used.     

Isolation and identification of a metabolite of cidofovir from rat kidney

Cidofovir is an acyclic nucleotide analog with potent and broad-spectrum antiviral activity against adenoviruses and herpesviruses including cytomegalovirus (CMV). Cidofovir undergoes intracellular phosphorylation by host enzymes to cidofovir phosphate and cidofovir diphosphate (the active form). An unidentified metabolite has been observed previously in rat tissues and in urine of rabbits, rats and monkeys dosed with cidofovir. In the present study, this metabolite was isolated from rat kidney following an intravenous dose of 100 mg kg⁻¹ cidofovir. The metabolite (metabolite I) was separated from cidofovir and impurities using extraction on anion-exchange resin followed by preparative normal and reversed-phase high-performance liquid chromatography (HPLC). The isolated metabolite I was subjected to proton, ¹³C and phosphorus nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization mass spectroscopy, and confirmed to be cidofovir–phosphocholine. The uptake of cidofovir by rat kidney was saturated at an intravenous dose of 100 mg kg⁻¹, probably as a result of saturation of the renal tubular secretion pathway. However, the relative abundance of cidofovir phosphocholine was not affected by dose.     

The application of LC-NMR and LC-MS for the separation and rapid structure elucidation of an unknown impurity in 5-aminosalicylic acid

This work has demonstrated the usefulness of combining liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR) and liquid chromatography–mass spectrometry (LC–MS) methodologies for a rapid identification of an unknown impurity (N1) in the drug 5-aminosalycilic acid. Complementary information obtained from the two methods has revealed plenty of structural information and led to the fast on-line structure determination of N1 prior to its isolation and purification. The analysis of LC–NMR and LC–MS spectra revealed that N1 and 5-aminosalycilic acid are structurally closely related compounds. The structure of N1 was later confirmed by high-resolution NMR spectroscopy of the isolated compound and the atom assignment was made. The approach described here has potential for 5-aminosalycilic acid impurity profiling and monitoring the production process.     

大鼠血浆中冬青素A的LC-MS测定法及其药动学研究(英文)

冬青素A系中药毛冬青的主要成分之一, 本文建立了液相色谱质谱法(LC-MS)研究冬青素A在大鼠体内的药动学特征。色谱分离采用C18柱, 甲醇-5 mM 醋酸铵(80:20, v/v) 为流动相质谱检测采用ESI源, 负离子检测, 冬青素A的检测离子为m/z 501.1→501.1,地高辛(内标) 的检测离子为m/z779.4→779.4。大鼠血浆加入磷酸溶液以乙酸乙酯提取, 分取有机层以氮气流吹干, 流动相复溶后进行LC-MS分析。方法学评价表明该法定量限为1.05 ng/mL, 在1.05-525.5 ng/mL范围内线性关系良好。日内和日间变异均小于10%, 提取回收率大于80%。采用建立的LC-MS法进行了大鼠单剂量口服冬青素A后, 其在大鼠体内的药动学研究, 获得了主要的药动学参数。     

A high-performance liquid chromatography and nuclear magnetic resonance spectroscopy-based analysis of commercially available praziquantel tablets

The amount of active ingredient in 20 commercially sourced batches of praziquantel (PZQ) tablets was determined using a high-performance liquid chromatography-ultraviolet (HPLC-UV) assay in conjunction with an anthentic, lot of PZQ powder. The general composition of each batch of tablets was also examined by means of (1)H nuclear magnetic resonance (NMR) spectroscopy and the NMR data were subjected to pattern recognition analysis by means of principal component analysis. The HPLC-UV results showed that each batch of PZQ tablets contained approximately the required amount of PZQ (600 mg per tablet). The NMR analysis showed a high degree of compositional variation between manufacturers, which caused by variation in excipients, along with some batch-to-batch variation in the tablets from a single manufacturer. Additionally, the PZQ tablets from one manufacturer were found to have an extra component (methyl-4-hydroxybenzoate) that was not detected in the other preparations.     

Structural elucidation of a PDE-5 inhibitor detected as an adulterant in a health supplement

A PDE-5 inhibitor was detected and isolated from a health supplement claimed to be a preparation of fresh oyster extracts and be able to promote and support healthy sexual function and endurance, etc. The structure of this PDE-5 inhibitor was elucidated using LC-UV, LC-TOF-MS, MS-MS, IR spectroscopy, and 2D NMR. It was characterized as 8-(2-(4-(hydroxymethyl)piperidin-1-yl)benzylamino)-3-ethyl-1H-imidazo[4,5-g]quinazoline-2(3H)-thione, a compound reported to be a PDE-5 inhibitor.     

液质联用法测定犬血浆中阿普唑仑及其代谢物α-羟基阿普唑仑的浓度

目的建立测定Beagle犬血浆中阿普唑仑及其代谢物α-羟基阿普唑仑浓度的液相色谱-质谱联用(LC-MS)法。方法血浆样品采用1 mol·L~(-1)硼酸盐缓冲液(pH 9.0)碱化、乙酸乙酯-正庚烷(85:15,V:V)萃取后LC-MS测定。色谱柱:Zorbax SB-C_(18)柱(150 mm×3 mm,3.5μm);流动相:乙腈-0.01 mol·L~(-1)乙酸胺缓冲液(含1%甲酸)(45:55,V:V);流速:0.3 mL·min~(-1);柱温:40℃。采用电喷雾正离子模式离子化,用于定量分析的离子分别为m/z 309.2(阿普唑仑)、m/z 325.2(α-羟基阿普唑仑)和m/z 343.2(三唑仑,内标)。结果阿普唑仑和α-羟基阿普唑仑的线性范围分别为0.5～50μg·L~(-1)和0.5～32μg·L~(-1),两者定量下限均为0.5μg·L~(-1),提取回收率均>80%,方法回收率为97.3%～102.5%,批内RSD≤10.4%,批间RSD≤12.2%。结论本方法灵敏、准确、重现性好,适用于阿普唑仑犬体内药动学研究。     

高脂饮食对阿雷地平及其活性代谢产物人体药动学特征的影响

目的评价高脂餐对阿雷地平及其活性代谢产物羟基阿雷地平在健康中国人体内药动学的影响。方法10名健康男性受试者空腹口服阿雷地平10 mg,经过1 wk清洗期后,受试者高脂餐后口服阿雷地平10 mg。采用液相色谱-串联质谱联用法测定血浆中阿雷地平及其活性代谢产物羟基阿雷地平的浓度。结果空腹和高脂餐后口服阿雷地平,阿雷地平的c_(max)分别为(2.4±s0.8)和(4.4±2.9)μg·L~(-1),羟基阿雷地平的c_(max)分别为(41±10)和(51±19)μg·L~(-1);空腹和高脂餐后阿雷地平和羟基阿雷地平的c_(max)无显著差异。空腹和高脂餐后口服阿雷地平,阿雷地平的AUC_(0～36)分别为(10.3±2.3)和(15±7)μg·h·L~(-1),羟基阿雷地平的AUC_(0～36)分别为(305±108)和(389±129)μg·h·L~(-1);阿雷地平的t_(max)分别为(4.4±1.0)和(9±6)h,羟基阿雷地平的f_(max)分别为(5.0±1.6)和(11±7)h;空腹和高脂餐后阿雷地平和羟基阿雷地平的AUC_(0～36)和t_(max)均存在显著差异,AUC显著增加,t_(max)显著延长。结论高脂餐后,阿雷地平的吸收出现延迟现象,吸收程度有所增加;羟基阿雷地平的体内生成延迟,但生成的量增加。     

Synthesis of the stable isotope labeled antiviral nucleoside analog [8‐13C–7,9‐15N2]‐ganciclovir

Ganciclovir, a nucleoside analog of 2′‐deoxyguanosine, is a drug used in suicide gene therapy for the treatment of mesothelioma. We required a stable isotope analog of ganciclovir for use in pharmacokinetic studies in order to monitor the systemic exposure of patients to the drug. Therefore, a facile and efficient synthesis of [8‐¹³C–7,9‐¹⁵N₂]‐ganciclovir, was devised. The synthesis was achieved in 4 steps with 25% total yield using commercially‐available [8‐¹³C–7,9‐¹⁵N₂]‐guanine, without the need for purification of intermediates. The key step of the synthesis involved the coupling of [8‐¹³C–7,9‐¹⁵N₂]‐guanine with 3‐propionyloxy‐2‐propionyloxy‐methoxypropyl propionate. The latter was synthesized from a commercially available dichlorohydrin. Each step of the reaction could be easily monitored by liquid chromatography–mass spectrometry. The structure of the labeled ganciclovir was confirmed using ¹H, ¹³C, and ¹⁵N nuclear magnetic resonance spectroscopy. Copyright © 2006 John Wiley & Sons, Ltd.     

Isolation, purification and identification of ellagic acid derivatives, catechins, and procyanidins from the root bark of Anisophyllea dichostyla R. Br.

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120g). The polyphenol content (3.32g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside and (-)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (-)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3'-di-O-methyl ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside, and 3'-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC-ESI-MS and nano-ESI-MS-MS: (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), epicatechin-(4beta-->8)-epicatechin (procyanidin B(2)), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-beta-d-glucopyranoside, (-)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4'-O- beta-d-glucopyranoside, and 3,3'-di-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (-)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC(50)=52 micro g/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3'-O-methyl ellagic acid 4'-O-beta-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), and (-)-epicatechin 3-O-gallate, proved to be the most potent (IC(50)=36 micro g/mL).     

Identification of benzamidenafil, a new class of phosphodiesterase-5 inhibitor, as an adulterant in a dietary supplement

A sample labeled to be a natural herbal supplement for the enhancement of sexual function, was sent to Health Sciences Authority (HSA) of Singapore for testing. An unknown compound was detected and isolated from the product. The structure of the unknown compound was identified using LC-UV, high-resolution MS, ESI-MS/MS, IR, and NMR. The compound was characterized as a phosphodiesterase-5 (PDE-5) inhibitor, benzamidenafil. This is the first report of benzamidenafil, representing a new class of PDE-5 inhibitors, as an adulterant of a dietary supplement.     

LC-MS/MS法同时测定人血浆中氯吡格雷及其羧酸代谢物的浓度

目的建立快速测定人血浆中氯吡格雷及其羧酸代谢物SR26334含量的LC-MS/MS法。方法乙醚-正己烷(4:1,V:V)2次液-液提取法(中性和酸化条件下),采用Teknokroma C_(18)色谱柱,以那格列奈和吡格列酮为内标,同时测定血浆中氯吡格雷和SR26334的浓度。流动相:甲醇-0.1%甲酸(80:20,V:V);流速:0.2mL·min~(-1);以多反应离子监测方式检测:氯吡格雷[M+H]~+,m/z 322.1→212.1;那格列奈[M+H]~+,m/z 318.3→166.2;氯吡格雷羧酸代谢物SR26334[M+H]~+,m/z 308.1→q98.1;吡格列酮[M+H]~+,m/z 357.2→134.2。结果氯吡格雷和内标那格列奈的保留时间分别在4.4和3.7min,SR26334和内标吡格列酮的保留时间分别在1.3和1.7min,氯吡格雷的线性范围为5～5000ng·L~(-1);SR26334的线性范围为20～2500μg·L~(-1)。提取回收率大于75%,方法回收率大于90%,日内、日间RSD小于10%(n=5)。结论本方法简便快速,适用于氧吡格雷制剂的新药临床研究和临床长期治疗病人血药浓度的常规监测。     

Validation of a New Sensitive Method for the Detection and Quantification of R and S-Epimers of Ergot Alkaloids in Canadian Spring Wheat Utilizing Deuterated Lysergic Acid Diethylamide as an Internal Standard

Ergot sclerotia effect cereal crops intended for consumption. Ergot alkaloids within ergot sclerotia are assessed to ensure contamination is below safety standards established for human and animal health. Ergot alkaloids exist in two configurations, the R and S-epimers. It is important to quantify both configurations. The objective of this study was to validate a new ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantification of six R and six S-epimers of ergot alkaloids in hard red spring wheat utilizing deuterated lysergic acid diethylamide (LSD-D₃) as an internal standard. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effects, recovery and precision were investigated. For the 12 epimers analyzed, low LOD and LOQ values were observed, allowing for the sensitive detection of ergot epimers. Matrix effects ranged between 101–113% in a representative wheat matrix. Recovery was 68.3–119.1% with an inter-day precision of <24% relative standard deviation (RSD). The validation parameters conform with previous studies and exhibit differences between the R and S-epimers which has been rarely documented. This new sensitive method allows for the use of a new internal standard and can be incorporated and applied to research or diagnostic laboratories.     

The metabolism of p-propylanisole in the rat and mouse and its variation with dose

We have studied the metabolism of the synthetic flavouring p-propylanisole in rats and mice and investigated the variation in its metabolism with dose. [methoxy-¹⁴C]-p-Propylanisole was given to female Wistar albino rats orally and male CD-1 mice ip at doses ranging from 0.05–1500 mg/kg body weight (0.2–20 μCi/animal). The urine, faeces and ¹⁴CO₂ in the expired air were collected. The urinary metabolites were separated by solvent extraction, thin-layer chromatography and high-pressure liquid chromatography, and characterized by mass and nuclear magnetic resonance spectroscopy and comparison with authentic samples. Three major ¹⁴C-labelled urinary metabolites were excreted, 1'- and 2'-hydroxy-p-propylanisole and p-methoxyhippuric acid. ¹⁴CO₂ was eliminated in the expired air, arising from oxidative O-demethylation. The relative quantities of the metabolites varied markedly with dose. The percentage of the dose that was O-demethylated fell as the dose increased and the proportion in the form of urinary metabolites increased. The relative proportions of the major urinary metabolites also changed with dose. In view of the great discrepancy between human exposure to p-propylanisole in foods (about 15 μg/day) and the doses used for its toxicological evaluation in animals, these results emphasize the importance of considering dose-dependent metabolism when interpreting the significance for man of animal data obtained at very high doses.     

Integrated HPLC-MS and 1H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides

1. Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics.

2. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-β-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an α,α-dimethylated ibuprofen analogue.

3. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37°C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and ¹H-NMR spectroscopy and the results compared.

4. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, ¹H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of ¹H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than ¹H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-β-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers.

5. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

     

The identification of a nitrosated prodrug of the PDE-5 inhibitor aildenafil in a dietary supplement: a Viagra with a pop

A new unapproved analogue of sildenafil was detected in capsules of a herbal dietary supplement promoted as a libido enhancing product. Using LC-DAD-MS, MS-MS, HRMS, IR and NMR the analogue was shown to be a derivative of the PDE-5 inhibitor aildenafil with a nitrosamine moiety. A hydrolysis experiment showed that the new analogue was a prodrug of aildenafil and was therefore named nitroso-prodenafil. A capsule contained 108 mg of nitroso-prodenafil which is equivalent to 84 mg of aildenafil and 5.1 mg of nitrogen monoxide (NO). Although it is unknown how much NO can be usefully generated there is 3-fold more NO present than in a 10 mg isorbide nitrate tablet. Both PDE-5 inhibitors and nitrosamines cause vasodilatation by increasing levels of NO. To their coincidental use is warned against because it may cause a fatal drop in blood pressure. In addition, nitrosamines are known carcinogens. This is the first time a PDE-5 inhibitor and a potential NO donor were identified in one molecule. The findings indicate the dangerous level of advancement in medicinal chemistry by producers of unapproved drugs.     

Prediction of designer drugs: Synthesis and spectroscopic analysis of synthetic cathinone analogs that may appear on the Swedish drug market

The use of hyphenated analytical techniques in forensic drug screening enables simultaneous identification of a wide range of different compounds. However, the appearance of drug seizures containing new substances, mainly new psychoactive substances (NPS), is steadily increasing. These new and other already known substances often possess structural similarities and consequently they exhibit spectral data with slight differences. This situation has made the criteria that ensure indubitable identification of compounds increasingly important. In this work, 6 new synthetic cathinones that have not yet appeared in any Swedish drug seizures were synthesized. Their chemical structures were similar to those of already known cathinone analogs of which 42 were also included in the study. Hence, a total of 48 synthetic cathinones making up sets of homologous and regioisomeric compounds were used to challenge the capabilities of various analytical techniques commonly applied in forensic drug screening, ie, gas chromatography–mass spectrometry (GC–MS), gas chromatography–Fourier transform infrared spectroscopy (GC–FTIR), nuclear magnetic resonance (NMR), and liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC–QTOF–MS). Special attention was paid to the capabilities of GC–MS and GC–FTIR to distinguish between the synthetic cathinones and the results showed that neither GC–MS nor GC–FTIR alone can successfully differentiate between all synthetic cathinones. However, the 2 techniques proved to be complementary and their combined use is therefore beneficial. For example, the structural homologs were better differentiated by GC–MS, while GC–FTIR performed better for the regioisomers. Further, new spectroscopic data of the synthesized cathinone analogs is hereby presented for the forensic community. The synthetic work also showed that cathinone reference compounds can be produced in few reaction steps.     

Characterisation and identification of the human N-glucuronide metabolite of cediranib

Cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline; RECENTIN™), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors, is currently in Phase III clinical trials for the first-line treatment of colorectal cancer and the treatment of recurrent glioblastoma.     

Application of metabonomic analytical techniques in the modernization and toxicology research of traditional Chinese medicine

In the recent years, a wide range of metabonomic analytical techniques are widely used in the modern research of traditional Chinese medicine (TCM). At the same time, the international community has attached increasing importance to TCM toxicity problems. Thus, many studies have been implemented to investigate the toxicity mechanisms of TCM. Among these studies, many metabonomic-based methods have been implemented to facilitate TCM toxicity investigation. At present, the most prevailing methods for TCM toxicity research are mainly single analysis techniques using only one analytical means. These techniques include nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), etc.; with these techniques, some favourable outcomes have been gained in the toxic reaction studies of TCM, such as the action target organs assay, the establishment of action pattern, the elucidation of action mechanism and the exploration of action material foundation. However, every analytical technique has its advantages and drawbacks, no existing analytical technique can be versatile. Multi-analysed techniques can partially overcome the shortcomings of single-analysed techniques. Combination of GC-MS and LC-MS metabolic profiling approaches has unravelled the pathological outcomes of aristolochic acid-induced nephrotoxicity, which can not be achieved by single-analysed techniques. It is believed that with the further development of metabonomic analytical techniques, especially multi-analysed techniques, metabonomics will greatly promote TCM toxicity research and be beneficial to the modernization of TCM in terms of extending the application of modern means in the TCM safety assessment, assisting the formulation of TCM safety norms and establishing the international standards indicators.     

Partial synthesis of five new analogues of the peptido-lactone Virginiamycine S1, modified in the fifth and/or sixth position ([Xxx5]-VS1 with Xxx = Ala,Asp, Asn and Lys and [Ala5,Gly6]-VS1)

We achieved the reconstruction of VS₁-analogues containing a substitute for the fifth residue, γ-oxo-Pip (Pip = pipecolic acid), starting from VS₁-pentapeptide (VS5P; 3) the latter being prepared by a two-step degradation process of the native antibiotic VS₁ ( 1a ). Protecting groups during the procedure were chosen in order to realize a minimal number of steps. Most of these gave excellent yields, including final cyclization between the fourth and fifth residue. In total, four analogues were synthesized with Ala, Asp, Asn and Lys ( 1b ) replacing γ-oxo-Pip. Among these, [Lys⁵(Tfa salt)]-VS₁ is water-soluble, which is an important characteristic for eventual application of VS₁ as a pharmaceutical agent. In the proposed reaction sequence, we made sure that residues 4 (MePhe) and 6 (Phg) became partially epimerised. We therefore obtained each time after cyclization a total of four epimers that have been separated by preparative TLC. The chiral identity of the final residues was realized by GC (Chirasil Val®-III) on the total hydrolysates.