Challenges in optimizing sample preparation and LC‐MS/MS conditions for the analysis of carglumic acid,an N‐acetyl glutamate derivative in human plasma

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed‐phase solid‐phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic‐lipophilic balance, and mixed‐mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed‐mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid‐13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) ? 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00‐6000 ng/mL (r² ≥ 0.9987). Matrix effect was evaluated in terms of IS‐normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra‐ and inter‐batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro and in vivo human metabolism of the synthetic cannabinoid AB‐CHMINACA

N‐[(1S)‐1‐(aminocarbonyl)‐2‐methylpropyl]‐1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamide (AB‐CHMINACA) is a recently introduced synthetic cannabinoid. At present, no information is available about in vitro or in vivo human metabolism of AB‐CHMINACA. Therefore, biomonitoring studies to screen AB‐CHMINACA consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated the in vitro metabolism of AB‐CHMINACA using human liver microsomes (HLMs). Formation of AB‐CHMINACA metabolites was monitored using liquid chromatography coupled to time‐of‐flight mass spectrometry. Twenty‐six metabolites of AB‐CHMINACA were detected including seven mono‐hydroxylated and six di‐hydroxylated metabolites and a metabolite resulting from N‐dealkylation of AB‐CHMINACA, all produced by cytochrome P450 (CYP) enzymes. Two carboxylated metabolites, likely produced by amidase enzymes, and five glucuronidated metabolites were also formed. Five mono‐hydroxylated and one carboxylated metabolite were likely the major metabolites detected. The involvement of individual CYPs in the formation of AB‐CHMINACA metabolites was tested using a panel of seven human recombinant CYPs (rCYPs). All the hydroxylated AB‐CHMINACA metabolites produced by HLMs were also produced by the rCYPs tested, among which rCYP3A4 was the most active enzyme. Most of the in vitro metabolites of AB‐CHMINACA were also present in urine obtained from an AB‐CHMINACA user, therefore showing the reliability of the results obtained using the in vitro metabolism experiments conducted to predict AB‐CHMINACA in vivo metabolism. The AB‐CHMINACA metabolites to target in biomonitoring studies using urine samples are now reliably identified and can be used for routine analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Using positron emission tomography to study human ketone body metabolism: A review

Ketone bodies – 3-hydroxybutyrate and acetoacetate – are important fuel substrates, which can be oxidized by most tissues in the body. They are synthesized in the liver and are derived from fatty acids released from adipose tissue. Intriguingly, under conditions of stress such as fasting, arterio-venous catheterization studies have shown that the brain switches from the use of almost 100% glucose to the use of > 50–60% ketone bodies. A similar adaptive mechanism is observed in the heart, where fasting induces a shift toward ketone body uptake that provides the myocardium with an alternate fuel source and also favorably affects myocardial contractility. Within the past years there has been a renewed interest in ketone bodies and the possible beneficial effects of fasting/semi-fasting/exercising and other “ketogenic” regimens have received much attention. In this perspective, it is promising that positron emission tomography (PET) techniques with isotopically labeled ketone bodies, fatty acids and glucose offer an opportunity to study interactions between ketone body, fatty acid and glucose metabolism in tissues such as the brain and heart. PET scans are non-invasive and thus eliminates the need to place catheters in vascular territories not easily accessible. The short half-life of e.g. 11C-labeled PET tracers even allows multiple scans on the same study day and reduces the total radiation burden associated with the procedure. This short review aims to give an overview of current knowledge on ketone body metabolism obtained by PET studies and discusses the methodological challenges and perspectives involved in PET ketone body research.     

川芎嗪通过抑制PI3K/Akt/mTOR通路诱导自噬改善糖尿病肾病大鼠肾损害

目的 探讨川芎嗪对糖尿病肾病（DN）大鼠肾脏 PI3K/Akt/mTOR信号通路和自噬标志蛋白 LC3B表达以及 尿微量白蛋白与尿肌酐比值（UACR）、肾脏病理的影响。方法 采用链脲佐菌素建立 DN大鼠模型,将模型大鼠随机 分为模型组,川芎嗪低、中、高剂量组,厄贝沙坦组;另设正常组,每组 12只。分别干预 8周后,采用酶法测定尿肌酐, 免疫比浊法测定尿微量白蛋白,计算 UACR;取肾组织,经甲醛固定后,进行苏木精-伊红（HE）和过碘酸-雪夫（PAS） 染色;通过蛋白免疫印迹法（Western blot）和免疫组化检测大鼠肾组织 PI3K/Akt/mTOR信号通路以及自噬标志蛋白 LC3B 表达的变化。结果 川芎嗪能减缓 DN 大鼠 UACR 的升高,改善其肾脏病理变化,其中川芎嗪中、高剂量组 UACR显著低于模型组（P＜0.05）,且川芎嗪中、高剂量组与厄贝沙坦组间比较差异无统计学意义（P＞0.05）。此外, 川芎嗪能抑制 DN大鼠肾组织 p-PI3K、p-Akt、p-mTOR的表达,进而提高自噬标志蛋白 LC3B的表达水平和 LC3B-Ⅱ/ LC3B-Ⅰ比值。结论 川芎嗪能降低 DN大鼠 UACR的升高、改善其肾脏病理变化,发挥以上肾保护作用的机制可能 与其抑制 PI3K/Akt/mTOR信号通路,进而促进肾脏自噬有关。     

两种微创术式治疗胆囊结石合并胆总管结石的应用体会

目的探讨腹腔镜胆囊切除胆总管探查术(LC-LBDE)与内镜下奥迪氏括约肌切开取石术(EST)联合腹腔镜胆囊切除术(LC)治疗胆囊结石合并胆总管结石的临床效果及术式选择。方法回顾分析我院2009年7月~2013年12月施行LC-LBDE 31例和EST+LC35例患者的临床资料,对手术时间、并发症、住院时间及住院费用等进行比较分析。结果两种治疗方式在手术疗效上无明显差异,并发症中LC-LBDE组发生率较高,住院费用相比,EST+LC组较高。结论对于胆囊结石合并胆总管结石的患者,上述两种手术方式均能达到有效的治疗目的,但是要根据不同的患者选择最合适的治疗方式。     

Pharmacokinetics of GHB and detection window in serum and urine after single uptake of a low dose of GBL – an experiment with two volunteers

During the last few years γ‐hydroxybutyric acid (GHB) and γ‐butyrolactone (GBL) have attracted much interest as recreational drugs and knock‐out drops in drug‐facilitated sexual assaults. This experiment aims at getting an insight into the pharmacokinetics of GHB after intake of GBL. Therefore Two volunteers took a single dose of 1.5 ml GBL, which had been spiked to a soft drink. Assuming that GBL was completely metabolized to GHB, the corresponding amount of GHB was 2.1 g. Blood and urine samples were collected 5 h and 24 h after ingestion, respectively. Additionally, hair samples (head hair and beard hair) were taken within four to five weeks after intake of GBL. Samples were analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) after protein precipitation with acetonitrile. The following observations were made: spiked to a soft drink, GBL, which tastes very bitter, formed a liquid layer at the bottom of the glass, only disappearing when stirring. Both volunteers reported weak central effects after approximately 15 min, which disappeared completely half an hour later. Maximum concentrations of GHB in serum were measured after 20 min (95 µg/ml and 106 µg/ml). Already after 4–5 h the GHB concentrations in serum decreased below 1 µg/ml. In urine maximum GHB concentrations (140 µg/ml and 120 µg/ml) were measured after 1–2 h, and decreased to less than 1 µg/ml within 8–10 h. The ratio of GHB in serum versus blood was 1.2 and 1.6. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid and simple LC‐MS/MS screening of 64 novel psychoactive substances using dried blood spots

The range of novel psychoactive substances (NPS) including phenethylamines, cathinones, piperazines, tryptamines, etc. is continuously growing. Therefore, fast and reliable screening methods for these compounds are essential and needed. The use of dried blood spots (DBS) for a fast straightforward approach helps to simplify and shorten sample preparation significantly. DBS were produced from 10 µl of whole blood and extracted offline with 500 µl methanol followed by evaporation and reconstitution in mobile phase. Reversed‐phase chromatographic separation and mass spectrometric detection (RP‐LC‐MS/MS) was achieved within a run time of 10 min. The screening method was validated by evaluating the following parameters: limit of detection (LOD), matrix effect, selectivity and specificity, extraction efficiency, and short‐term and long‐term stability. Furthermore, the method was applied to authentic samples and results were compared with those obtained with a validated whole blood method used for routine analysis of NPS. LOD was between 1 and 10 ng/ml. No interference from matrix compounds was observed. The method was proven to be specific and selective for the analytes, although with limitations for 3‐FMC/flephedrone and MDDMA/MDEA. Mean extraction efficiency was 84.6 %. All substances were stable in DBS for at least a week when cooled. Cooling was essential for the stability of cathinones. Prepared samples were stable for at least 3 days. Comparison to the validated whole blood method yielded similar results. DBS were shown to be useful in developing a rapid screening method for NPS with simplified sample preparation. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of potential metabolic biomarkers in Yin deficiency syndrome using LC–MS

Yin and Yang are the two counter-balancing aspects in ancient Chinese philosophy. In traditional Chinese medicine, Yin deficiency syndrome (YDS) is a common sub-health state with complex causes. While the syndrome may be treated to various degrees of effectiveness with traditional Chinese medicine, efficient modern methods are yet to be developed for diagnosing and treating the YDS. Here we performed a metabolomics study on YDS in rats. Serum metabolites in rats were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC–MS) method to identify potential biomarkers for YDS. The rats were divided randomly into the healthy control group, the untreated YDS group, and the anemarrhena treated YDS group. Compared with the control group, significant increase in the metabolites such as dihydrotestosterone (DHT) and 5β-DHT, 4-imidazolone-5-propanoate, 4-(L-alanin-3-yl)-2-hydroxy-cis,cis-muconate 6-semialdehyde, and 5-(L-alanin-3-yl)-2-hydroxy-cis,cis-muconate 6-semialdehyde were observed in the serum of untreated YDS group, which returned to normal in the anemarrhena treated group. Therefore, these metabolites may serve as potential biomarkers for YDS, and may facilitate the diagnosis and treatment of YDS.     

Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.