Selective HPLC Method Development for Soy Phosphatidylcholine Fatty Acids and Its Mass Spectrometry

A novel, efficient and simple approach for soy phosphatidylcholine analysis according to its fatty acid composition was studied with reverse-phase high-performance liquid chromatography. The reverse-phase high-performance liquid chromatography analysis was performed isocratically using UV detector and simple mobile phase solvents consisting of isopropyl alcohol, methanol, and deionized water in the proportion of 70:8:22 v/v. The uniqueness of the proposed method was the separation of individual fatty acids of soy phosphatidylcholine. The high-performance liquid chromatography method for soy phosphatidylcholine was validated for linearity with correlation coefficient of above 0.99 for all the peaks separated according to their fatty acid composition. The intra-day and the inter-day precision studies provided the relative standard deviation of less than 2%. The limit of detection and limit of quantitation values were also calculated for all the resolved peaks of soy phosphatidylcholine. Also system performance parameters such as number of theoretical plates, capacity factor, tailing factor, separation factor, and peak resolution were studied systematically and found well within the acceptable range. The proposed high-performance liquid chromatography method was successfully applied to soy phosphatidylcholine extracted and purified from deoiled soy lecithin without any interference of impurities or solvent peaks. Individually, the collected peaks of sample soy phosphatidylcholine were subjected for mass spectroscopy. The mass spectra showed all the peaks having different saturated or unsaturated fatty acid chains attached to glyerophosphocholine moiety of soy phosphatidylcholine. The method developed is economic and well suited for estimation of soy phosphatidylcholine with its fatty acid composition.     

Generation of microbubbles for diagnostic and therapeutic applications using a novel device

Developments in both diagnostic and therapeutic applications of microbubbles have greatly increased the need for more advanced preparation technologies which provide a well-defined, narrow microbubble size-distribution. In this paper, we demonstrate the use of a new device, consisting of a T-junction whose outlet capillary is fitted with an electrohydrodynamic spraying arrangement, to prepare phospholipid-coated air microbubbles, making significant advances in controlling and decreasing the size and size-distribution, and increasing the stability/lifetime of the bubbles prepared. The microbubbles were characterised via optical microscopy to determine the relationship between the size-distribution obtained and the process variables, specifically the flow rates of the phospholipid suspension and air (Q_l and Q_g), and the applied voltage (V). The formation of microbubbles in the device was also studied using high-speed photography. For the range of parameters investigated, the bubble diameter was found to scale with the product of the flow rate ratio (Q_l/Q_g) and the applied voltage, with a consistent bubble diameter of 5.1 ± 2 μm being obtained at Q_l/Q_g = 1.7 and V = 18 kV. The bubbles prepared using this method were found to be stable for at least 2 h at ambient temperature and pressure.     

Characterization of the Percutaneous Absorption of Ketoprofen Using the Franz Skin Finite Dose Model

Objectives: Ketoprofen is an analgesic, anti-inflammatory agent commonly used in the management of chronic musculoskeletal pain. The aim of this study is to characterize the percutaneous absorption of 2 ketoprofen formulations (ketoprofen 10% in Pluronic Lecithin Organogel (PLO) and ketoprofen 10% in Lipoderm, referred to as phospholipid base), when applied to the human cadaver trunk skin, in vitro, using the Franz skin finite dose model. PLO and phospholipid base are vehicles used to facilitate the delivery of drugs into and through the skin following topical applications.

Methods: The percutaneous absorption of ketoprofen was evaluated using human cadaver trunk skin from 3 donors. The skin was cut into small sections and cultured within Franz diffusion cells. A variable finite dose of each formulation was then applied to 3 replicate skin sections per donor and receptor solutions were collected at predetermined time points (0, 4, 8, 12, 24, 32, and 48 hr). After the last receptor sample was collected, skin surfaces were washed and split into epidermis and dermis. Collected samples were analyzed using HPLC.

Results: Both PLO and phospholipid base were capable of facilitating the absorption of ketoprofen across human cadaver trunk skin. However, ketoprofen, when in phospholipid base, showed higher mean total absorption (p = 0.022) and faster rate of absorption (p < 0.05 at 2, 6, 10, and 18 hr) than when in PLO.

Conclusion: Chronic musculoskeletal pain can be a major burden for most patients, affecting their lifestyle and reducing overall quality of life. When compared to PLO, phospholipid base has the ability to potentially deliver higher concentrations of ketoprofen to underlying soft tissues and at a more rapid rate. With more ketoprofen at the site of injury, the analgesic and anti-inflammatory effects will likely be enhanced, potentially reducing pain and improving quality of life.     

Measurement of Circulating Phospholipid Fatty Acids: Association between Relative Weight Percentage and Absolute Concentrations

Objective: Most epidemiologic studies of circulating phospholipid fatty acids (PLFAs) and disease risk have used the relative concentration (percentage of total) of each fatty acid as the measure of exposure. Using relative concentrations, the total of all fatty acids is summed to 100% and thus the values of individual fatty acid are not independent. This has led to debate, along with the suggestion to use absolute concentrations of fatty acids. We aimed to examine the relationship between relative (weight percentage) and absolute (mg/L) concentrations of individual circulating PLFAs.

Methods: Relative and absolute concentrations of 41 circulating PLFAs were measured by gas chromatography in samples from 3 diverse populations. Correlations between the relative and absolute concentrations for each fatty acid were used to measure agreement. Unadjusted correlations and correlations adjusting absolute PLFA concentrations for total cholesterol were calculated.

Results: Unadjusted correlations between relative and absolute concentrations, as well as correlations adjusting absolute PLFA concentrations for total cholesterol, were high for most PLFAs in all 3 studies. Across the 3 studies, 28 of the 41 analyzed PLFAs had unadjusted correlations > 0.6 and 39 had adjusted correlations > 0.6.

Conclusions: Choice of relative vs absolute concentration may not affect interpretation of results for most circulating PLFAs in studies of association between individual PLFAs and disease outcomes, especially if a covariate reflecting total lipids, such as total circulating cholesterol, is included in the model. However, for fatty acids, such as 16:0 (palmitic acid), with low correlation between the 2 metrics, using relative vs absolute concentration may lead to different inferences regarding their association with the outcome. Because both concentrations could be obtained simultaneously from the same laboratory assay, use of both metrics is warranted to better understand PLFA–disease relationships.     

匹多莫德磷脂复合物的制备及鉴别

目的 制备匹多莫德磷脂复合物并对复合物进行鉴别。方法 以结合率为评价标准,结合单因素考察与正交试验设计法确定匹多莫德磷脂复合物的制备方法,并采用红外分析法、差式扫描量热分析、X-射线衍射分析鉴别所制得的复合物。结果 匹多莫德磷脂复合物反应条件如下：乙醇为反应溶剂,匹多莫德与磷脂的投料摩尔比为1：1,反应液中药物浓度为8 mg·mL^-1,于60℃下磁力搅拌1 h。复合物红外图谱发生变化;差热扫描显示复合物的相变温度改变;X-射线衍射分析显示复合物呈现无定型特征。结论 确定制备匹多莫德磷脂复合物的最佳工艺,匹多莫德与磷脂形成复合物,结构不同于单体及物理混合物。     

A subchronic toxicity study of Radix Dipsaci water extract by oral administration in F344 rats

Radix Dipsaci, the dried root of Dipsacus asperoides C.Y. Cheng & T.M.Ai, has therapeutic effects on various disorders, and in particular, bone and joint disease. Despite such ethnomedicinal benefits, there is very little information regarding its in vivo toxicity or adverse effects. This study was conducted to evaluate the potential toxicity of the Radix Dipsaci water Extract (RD-wE) by using F344 rats. The RD-wE was administered orally to rats at doses of 0, 125, 250, 500, 1000, and 2000 mg/kg body weight (bw)/day for 13 weeks. During the treatment period there were no mortalities attributed to RD-wE. Moreover, no toxic effects were observed with regard to body weight, clinical pathology (hematology, clinical biochemistry, and urinalysis), and anatomic pathology (gross findings, organ weight, and microscopic examination). The changes related to the treatment were excessive salivation at the mouth and soft feces, observed in male and female rats at 1000 or 2000 mg/kg bw/day, but these were not accompanied by any microscopic correlate or other pathophysiological changes. Based on these results, the oral no-observed-adverse-effect level of the RD-wE was considered to be 2000 mg/kg bw/day in both genders, although the target organs were not determined under the current experimental conditions.     

三叶豆紫檀苷磷脂复合物自微乳研制及跨膜转运研究

目的从苦参中大量制备三叶豆紫檀苷(trifolirhizin,Tri),通过制成磷脂复合物自微乳改善Tri的吸收。方法采用硅胶柱色谱、重结晶等技术分离制备Tri,综合理化性质和波谱学数据进行结构鉴定。分别采用正交试验、星点设计效应面法优化Tri磷脂复合物(TPC)、TPC自微乳(TPC-SMEDDS)处方。Caco-2模型考察Tri、TPC及TPC-SMEDDS的跨膜转运。结果磷脂复合物的反应物(即药物Tri)质量浓度为4 mg/m L,Tri与卵磷脂投料物质的量比为1∶1.5,反应时间为3 h,复合率可达(93.20±2.01)%。优化的自微乳油相为丙二醇单辛酸酯(Capyrol 90),质量分数占43.65%;乳化剂选择聚氧乙烯蓖麻油(Cremphor EL40),助乳化剂为二乙二醇单乙基醚(Transcutol HP),二者质量比(Km)为7.58。Caco-2细胞实验表明,Tri的表观渗透系数(Papp)为2.45×10-7 cm/s;TPC及TPC-SMEDDS的Papp分别为5.13×10-6 cm/s和1.847×10-5 cm/s。结论磷脂复合物及自微乳技术联用可以明显改善Tri的渗透系数,提高Tri跨膜转运效率,提示可提高生物利用度。     

Nanosized ethosomes-based hydrogel formulations of methoxsalen for enhanced topical delivery against vitiligo: formulation optimization,in vitro evaluation and preclinical assessment

The present investigation aimed for the development and characterization of ethosomes-based hydrogel formulations of methoxsalen for enhanced topical delivery and effective treatment against vitiligo. The ethosomes were prepared by central composite design (CCD) and characterized for various quality attributes like vesicle shape, size, zeta potential, lamellarity, drug entrapment and drug leaching. The optimized ethosomes were subsequently incorporated int Carbopol® 934 gel and characterized for drug content, rheological behavior, texture profile, in vitro release, ex vivo skin permeation and retention, skin photosensitization and histopathological examination. Ethosomes were found to be spherical and multilamellar in structures having nanometric size range with narrow size distribution, and high encapsulation efficiency. Ethosomal formulations showed significant skin permeation and accumulation in the epidermal and dermal layers. The fluorescence microscopy study using 123 Rhodamine exhibited enhanced permeation of the drug-loaded ethosomes in the deeper layers of skin. Also, the developed formulation showed insignificant phototoxicity and erythema vis-à-vis the conventional cream. The results were cross-validated using histopathological examination of skin segments. In a nutshell, the ethosomes-based hydrogel formulation was found to be a promising drug delivery system demonstrating enhanced percutaneous penetration of methoxsalen with reduced phototoxicity and erythema, thus leading to improved patient compliance for the treatment against vitiligo.     

Analysis of pulmonary surfactant in rat lungs after inhalation of nanomaterials: Fullerenes,nickel oxide and multi-walled carbon nanotubes

The health risks of inhalation exposure to engineered nanomaterials in the workplace are a major concern in recent years, and hazard assessments of these materials are being conducted. The pulmonary surfactant of lung alveoli is the first biological entity to have contact with airborne nanomaterials in inhaled air. In this study, we retrospectively evaluated the pulmonary surfactant components of rat lungs after a 4-week inhalation exposure to three different nanomaterials: fullerenes, nickel oxide (NiO) nanoparticles and multi-walled carbon nanotubes (MWCNT), with similar levels of average aerosol concentration (0.13–0.37?mg/m³). Bronchoalveolar lavage fluid (BALF) of the rat lungs stored after previous inhalation studies was analyzed, focusing on total protein and the surfactant components, such as phospholipids and surfactant-specific SP-D (surfactant protein D) and the BALF surface tension, which is affected by SP-B and SP-C. Compared with a control group, significant changes in the BALF surface tension and the concentrations of phospholipids, total protein and SP-D were observed in rats exposed to NiO nanoparticles, but not in those exposed to fullerenes. Surface tension and the levels of surfactant phospholipids and proteins were also significantly different in rats exposed to MWCNTs. The concentrations of phospholipids, total protein and SP-D and BALF surface tension were correlated significantly with the polymorphonuclear neutrophil counts in the BALF. These results suggest that pulmonary surfactant components can be used as measures of lung inflammation.     

In vivo brain (31)P-MRS: measuring the phospholipid resonances at 4 Tesla from small voxels

An optimized phosphorous ((31)P) three-dimensional chemical-shift imaging (3D-CSI) protocol was developed at 4 T to study the phospholipid metabolism from discrete regions in the human brain without the need for (1)H-decoupling or nuclear Overhauser enhancement (NOE). In this study, a spherically bound, weighted average, random point omission 3D-CSI technique was developed and tested, based on methods proposed in the literature. The technique yields a significant (p < 0.001, two-tailed, 5% confidence level) increase in signal-to-noise (SNR) efficiency over conventional 3D-CSI (phantom 32%), without an increase in voxel bleedthrough. An automated time-domain fitting procedure utilizing prior spectral knowledge quantified the individual brain phospholipid metabolites from 15 cm(3) effective (8.0 cm(3) nominal) volumes from the left/right-parieto-occipital cortex and left/right thalamus in 10 normal volunteers. Individual constituents from the phosphomonoester (PME) region; phosphoethanolamine (PEth), phosphocholine (PCh) and the phosphodiester (PDE) region; glycerophosphoethanolamine (GPEth), glycerophosphocholine (GPCh) and membrane phospholipids (MP) were separately quantified to assess the precision of our method at 4 T against previous (1)H-decoupled (31)P-MRS brain studies at lower fields and much larger voxels. Derived concentrations (mM/l tissue) for PEth, PCh, GPEth, GPCh and MP in the left-parieto-occipital cortex were 0.81 +/- 0.21, 0.46 +/- 0.14, 0.74 +/- 0.30, 1.15 +/- 0.43 and 1.54 +/- 0.95 mM, respectively, and 0.94 +/- 0.16, 0.46 +/- 0.17, 0.83 +/- 0.22, 1.14 +/- 0.40 and 1.26 +/- 0.78 mM for the right parieto-occipital cortex. Derived concentrations (mM/l tissue) for PEth, PCh, GPEth, GPCh and MP in the left-thalamus were 0.69 +/- 0.18, 0.42 +/- 0.16, 0.63 +/- 0.20, 1.05 +/- 0.42 and 0.93 +/- 0.56 mM, respectively, and 0.68 +/- 0.24, 0.34 +/- 0.18, 0.60 +/- 0.23, 1.09 +/- 0.36 and 0.74 +/- 0.48 mM for the right-thalamus. This is the first study to our knowledge that has been able to quantify each of these individual phospholipid metabolites from such small voxels in the brain within a clinically reasonable scan time and without (1)H-decoupling or NOE.