Precision Nutrition and Cardiovascular Disease Risk Reduction: the Promise of High-Density Lipoproteins.

PURPOSE OF REVIEW: Emerging evidence supports the promise of precision nutritional approaches for cardiovascular disease (CVD) prevention. Here, we discuss current findings from precision nutrition trials and studies reporting substantial inter-individual variability in responses to diets and dietary components relevant to CVD outcomes. We highlight examples where early precision nutrition research already points to actionable intervention targets tailored to an individual's biology and lifestyle. Finally, we make the case for high-density lipoproteins (HDL) as a compelling next generation target for precision nutrition aimed at CVD prevention. HDL possesses complex structural features including diverse protein components, lipids, size distribution, extensive glycosylation, and interacts with the gut microbiome, all of which influence HDL's anti-inflammatory, antioxidant, and cholesterol efflux properties. Elucidating the nuances of HDL structure and function at an individual level may unlock personalized dietary and lifestyle strategies to optimize HDL-mediated atheroprotection and reduce CVD risk. RECENT FINDINGS: Recent human studies have demonstrated that HDL particles are key players in the reduction of CVD risk. Our review highlights the role of HDL and the importance of personalized therapeutic approaches to improve their potential for reducing CVD risk. Factors such as diet, genetics, glycosylation, and gut microbiome interactions can modulate HDL structure and function at the individual level. We emphasize that fractionating HDL into size-based subclasses and measuring particle concentration are necessary to understand HDL biology and for developing the next generation of diagnostics and biomarkers. These discoveries underscore the need to move beyond a one-size-fits-all approach to HDL management. Precision nutrition strategies that account for personalized metabolic, genetic, and lifestyle data hold promise for optimizing HDL therapies and function to mitigate CVD risk more potently. While human studies show HDL play a key role in reducing CVD risk, recent findings indicate that factors such as diet, genetics, glycosylation, and gut microbes modulate HDL function at the individual level, underscoring the need for precision nutrition strategies that account for personalized variability to optimize HDL's potential for mitigating CVD risk.

A single 36-h water-only fast vastly remodels the plasma lipidome.

Background: Prolonged fasting, characterized by restricting caloric intake for 24 h or more, has garnered attention as a nutritional approach to improve lifespan and support healthy aging. Previous research from our group showed that a single bout of 36-h water-only fasting in humans resulted in a distinct metabolomic signature in plasma and increased levels of bioactive metabolites, which improved macrophage function and lifespan in C. elegans. Objective: This secondary outcome analysis aimed to investigate changes in the plasma lipidome associated with prolonged fasting and explore any potential links with markers of cardiometabolic health and aging. Method: We conducted a controlled pilot study with 20 male and female participants (mean age, 27.5 ± 4.4 years; mean BMI, 24.3 ± 3.1 kg/m2) in four metabolic states: (1) overnight fasted (baseline), (2) 2-h postprandial fed state (fed), (3) 36-h fasted state (fasted), and (4) 2-h postprandial refed state 12 h after the 36-h fast (refed). Plasma lipidomic profiles were analyzed using liquid chromatography and electrospray ionization mass spectrometry. Results: Several lipid classes, including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylethanolamine, and triacylglycerol were significantly reduced in the 36-h fasted state, while free fatty acids, ceramides, and sphingomyelin were significantly increased compared to overnight fast and fed states (P < 0.05). After correction for multiple testing, 245 out of 832 lipid species were significantly altered in the fasted state compared to baseline (P < 0.05). Random forest models revealed that several lipid species, such as LPE(18:1), LPC(18:2), and FFA(20:1) were important features in discriminating the fasted state from both the overnight fasted and postprandial state. Conclusion: Our findings indicate that prolonged fasting vastly remodels the plasma lipidome and markedly alters the concentrations of several lipid species, which may be sensitive biomarkers of prolonged fasting. These changes in lipid metabolism during prolonged fasting have important implications for the management of cardiometabolic health and healthy aging, and warrant further exploration and validation in larger cohorts and different population groups.

Seasonal Factors Are Associated with Activities of Enzymes Involved in High-Density Lipoprotein Metabolism among Pregnant Females in Ghana.

Background: Small-quantity lipid-based nutrient supplements (SQ-LNS) during pregnancy and postnatally were previously shown to improve high-density lipoprotein (HDL) cholesterol efflux capacity (CEC) and length in the children of supplemented mothers at 18 mo of age in the International Lipid-Based Nutrient Supplements (iLiNS) DYAD trial in Ghana. However, the effects of SQ-LNS on maternal HDL functionality during pregnancy are unknown. Objective: The goal of this cross-sectional, secondary outcome analysis was to compare HDL function in mothers supplemented with SQ-LNS vs. iron and folic acid (IFA) during gestation. Methods: HDL CEC and the activities of 3 HDL-associated enzymes were analyzed in archived plasma samples (N = 197) from a subsample of females at 36 weeks of gestation enrolled in the iLiNS-DYAD trial in Ghana. Correlations between HDL function and birth outcomes, inflammatory markers C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP), and the effects of season were explored to determine the influence of these factors on HDL function in this cohort of pregnant females. Results: There were no statistically significant differences in HDL CEC, plasma lecithin-cholesterol acyltransferase (LCAT) activity, cholesteryl ester transfer protein (CETP) activity, or phospholipid transfer protein (PLTP) activity between mothers supplemented with SQ-LNS compared with IFA control, and no statistically significant relationships between maternal HDL function and childbirth outcomes. LCAT activity was negatively correlated with plasma AGP (R = -0.19, P = 0.007) and CRP (R = -0.28, P < 0.001), CETP and LCAT activity were higher during the dry season compared to the wet season, and PLTP activity was higher in the wet season compared to the dry season. Conclusions: Mothers in Ghana supplemented with SQ-LNS compared with IFA during gestation did not have measurable differences in HDL functionality, and maternal HDL function was not associated with childbirth outcomes. However, seasonal factors and markers of inflammation were associated with HDL function, indicating that these factors had a stronger influence on HDL functionality than SQ-LNS supplementation during pregnancy. Clinical Trial Registry number: The study was registered as NCT00970866. https://clinicaltrials.gov/study/NCT00970866.

The Advantage of Proton Therapy in Hypothalamic-Pituitary Axis and Hippocampus Avoidance for Children with Medulloblastoma.

PURPOSE: Craniospinal irradiation (CSI) improves clinical outcomes at the cost of long-term neuroendocrine and cognitive sequelae. The purpose of this pilot study was to determine whether hypothalamic-pituitary axis (HPA) and hippocampus avoidance (HPA-HA) with intensity-modulated proton therapy (IMPT) can potentially reduce this morbidity compared with standard x-ray CSI. MATERIALS AND METHODS: We retrospectively evaluated 10 patients with medulloblastoma (mean, 7 years; range, 4-14 years). Target volumes and organs at risk were delineated as per our local protocol and the ACNS0331 atlas. An experienced neuroradiologist verified the HPA and hippocampus contours. The primary objective was CSI and boost clinical target volume (CTV) covering 95% of the volume (D95) > 99% coverage with robustness. Described proton therapy doses in grays are prescribed using a biological effectiveness relative to photon therapy of 1.1. The combined prescribed dose in the boost target was 54 Gy. Secondary objectives included the HPA and hippocampus composite average dose (Dmean ≤ 18 Gy). For each patient, volumetric modulated arc radiotherapy (VMAT) and tomotherapy (TOMO) plans existed previously, and a new plan was generated with 3 cranial and 1 or 2 spinal beams for pencil-beam scanning delivery. Statistical comparison was performed with 1-way analysis of variance. RESULTS: Compared with standard CSI, HPA-HA CSI had statistically significant decreases in the composite doses received by the HPA (32.2 versus 17.9 Gy; P < .001) and hippocampi (39.8 versus 22.8 Gy; P < .001). The composite HPA Dmean was lower in IMPT plans (17.9 Gy) compared with that of VMAT (21.8 Gy) and TOMO (21.2 Gy) plans (P = .05). Hippocampi composite Dmean was also lower in IMPT plans (21 Gy) compared with that of VMAT (27.5 Gy) and TOMO (27.2 Gy) plans (P = .02). The IMPT CTV D95 coverage was lower in IMPT plans (52.8 Gy) compared with that of VMAT (54.6 Gy) and TOMO (54.6 Gy) plans (P < .001) The spared mean volume was only 1.35% (19.8 cm3) of the whole-brain CTV volume (1476 cm3). CONCLUSION: We found that IMPT has the strong potential to reduce the dose to the HPA and hippocampus, compared with standard x-ray CSI while maintaining target coverage. A prospective clinical trial is required to establish the safety, efficacy, and toxicity of this novel CSI approach.

Multi-Omic Analyses Reveal Bifidogenic Effect and Metabolomic Shifts in Healthy Human Cohort Supplemented With a Prebiotic Dietary Fiber Blend.

Dietary fiber, a nutrient derived mainly from whole grains, vegetables, fruits, and legumes, is known to confer a number of health benefits, yet most Americans consume less than half of the daily recommended amount. Convenience and affordability are key factors determining the ability of individuals to incorporate fiber-rich foods into their diet, and many Americans struggle to access, afford, and prepare foods rich in fiber. The objective of this clinical study was to test the changes in microbial community composition, human metabolomics, and general health markers of a convenient, easy to use prebiotic supplement in generally healthy young participants consuming a diet low in fiber. Twenty healthy adults participated in this randomized, placebo-controlled, double-blind, crossover study which was registered at clinicaltrials.gov as NCT03785860. During the study participants consumed 12 g of a prebiotic fiber supplement and 12 g of placebo daily as a powder mixed with water as part of their habitual diet in randomized order for 4 weeks, with a 4-week washout between treatment arms. Fecal microbial DNA was extracted and sequenced by shallow shotgun sequencing on an Illumina NovaSeq. Plasma metabolites were detected using liquid chromatography-mass spectrometry with untargeted analysis. The phylum Actinobacteria, genus Bifidobacterium, and several Bifidobacterium species (B. bifidum, B. adolescentis, B. breve, B. catenulatum, and B. longum) significantly increased after prebiotic supplementation when compared to the placebo. The abundance of genes associated with the utilization of the prebiotic fiber ingredients (sacA, xfp, xpk) and the production of acetate (poxB, ackA) significantly changed with prebiotic supplementation. Additionally, the abundance of genes associated with the prebiotic utilization (xfp, xpk), acetate production (ackA), and choline to betaine oxidation (gbsB) were significantly correlated with changes in the abundance of the genus Bifidobacterium in the prebiotic group. Plasma concentrations of the bacterially produced metabolite indolepropionate significantly increased. The results of this study demonstrate that an easy to consume, low dose (12 g) of a prebiotic powder taken daily increases the abundance of beneficial bifidobacteria and the production of health-promoting bacteria-derived metabolites in healthy individuals with a habitual low-fiber diet. Clinical Trial Registration: www.clinicaltrials.gov/, identifier: NCT03785860.

Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Functional cranio-spinal irradiation: A hippocampal and hypothalamic-pituitary axis sparing radiation technique using two IMRT modalities.

Cranio-spinal irradiation (CSI) treatment of embryonal tumors is associated with long-term endocrine and neuro-cognitive sequelae. As an example, the radiation regiment for standard risk medulloblastoma is 23.4 Grays (Gy) CSI followed by a boost of 30.6Gy to the tumor bed. We hypothesize that a novel CSI technique, which we named "Functional" CSI (F-CSI) can reduce the dose to the hypothalamic-pituitary axis (HPA) and hippocampi compared to standard CSI (S-CSI) without sacrificing coverage. In this study, we compared the efficacy of Volumetric Modulated Arc Therapy (VMAT) and Helical Tomotherapy (HT) in delivering this novel CSI technique. Plans were constructed from 10 patients with embryonal tumors previously treated at our institution. Target volumes and organs at risk were delineated as per our local protocol and the ACNS0331 Atlas. The HPA and hippocampi contours were verified by an experienced neuro-radiologist. Primary objective was to achieve a D95% to the prescribed dose of 23.4Gy for CSI and 30.6Gy for the boost. Dmean ≤18Gy was assigned to the HPA and hippocampi. A two-sided t-test was used for comparison. F-CSI in both modalities were able to achieve the D95% target coverage. Hot spots (D2%) were lower with HT for both the CSI component (p = 0.03) and boost component (p < 0.01). VMAT was able to achieve better conformality (p < 0.01). Compared to S-CSI, both F-CSI modalities were able to achieve a significant decrease in dose to the HPA and Hippocampi. The average S-CSI HPA and Hippocampi Dmean were 23.9Gy and 23.8Gy. In contrast, respective F-CSI Dmean were 13.9Gy and 17.2Gy in VMAT and 15Gy and 15.9Gy in HT. The average composite (F-CSI plus boost) Dmean to the HPA and hippocampi often exceeded 18Gy. Compared to S-CSI, F-CSI with VMAT and HT were capable of achieving acceptable coverage while sparing the HPA and hippocampi. However, the addition of the boost component often exceeded the mean dose of 18Gy. This may be overcome with more conformal modalities for the boost phase such as stereotactic radiotherapy or proton therapy.

A Guide to Diet-Microbiome Study Design.

Intense recent interest in understanding how the human gut microbiome influences health has kindled a concomitant interest in linking dietary choices to microbiome variation. Diet is known to be a driver of microbiome variation, and yet the precise mechanisms by which certain dietary components modulate the microbiome, and by which the microbiome produces byproducts and secondary metabolites from dietary components, are not well-understood. Interestingly, despite the influence of diet on the gut microbiome, the majority of microbiome studies published to date contain little or no analysis of dietary intake. Although an increasing number of microbiome studies are now collecting some form of dietary data or even performing diet interventions, there are no clear standards in the microbiome field for how to collect diet data or how to design a diet-microbiome study. In this article, we review the current practices in diet-microbiome analysis and study design and make several recommendations for best practices to provoke broader discussion in the field. We recommend that microbiome studies include multiple consecutive microbiome samples per study timepoint or phase and multiple days of dietary history prior to each microbiome sample whenever feasible. We find evidence that direct effects of diet on the microbiome are likely to be observable within days, while the length of an intervention required for observing microbiome-mediated effects on the host phenotype or host biomarkers, depending on the outcome, may be much longer, on the order of weeks or months. Finally, recent studies demonstrating that diet-microbiome interactions are personalized suggest that diet-microbiome studies should either include longitudinal sampling within individuals to identify personalized responses, or should include an adequate number of participants spanning a range of microbiome types to identify generalized responses.

Variant CYP2C9 alleles and warfarin concentrations in patients receiving low-dose versus average-dose warfarin therapy.

This study compared the frequency of variant cytochrome P450 2C9 (CYP2C9) alleles and warfarin S/R concentration ratio in patients who required low-dose (<2.5 mg/day) and average-dose (5+/-0.5 mg/day) warfarin. Patients who achieved a therapeutic international normalized ratio were recruited from the Atlanta Veterans Affairs Medical Center anticoagulation clinic. CYP2C9*2 and *3 alleles were determined by validated Taqman allelic discrimination assays. Warfarin S and R concentrations were determined by chiral capillary electrochromatography with electrospray ionization mass spectrometry. At least 1 variant allele was found in 66.7% and 22.2% of patients in the low-dose and average-dose groups, respectively (P= .001, chi(2)). The warfarin S/R concentration ratio was 0.665 (range, 0.162-3.58) and 0.452 (range, 0.159-2.36) for patients receiving low-dose and average-dose therapy, respectively (P= .097). A warfarin requirement of <2.5 mg/day and an elevated warfarin S/R concentration ratio were each associated with a higher frequency of variant CYP2C9 alleles.

Lack of pharmacokinetic interaction between lamotrigine and olanzapine in healthy volunteers.

STUDY OBJECTIVE: To investigate the potential drug-drug interaction between lamotrigine, an antiepileptic agent used to treat bipolar disorders, and olanzapine, an atypical antipsychotic drug also used to treat bipolar disorders, both of which are metabolized by the uridine diphosphate glucuronosyltransferase system. DESIGN: Prospective cohort study. SETTING: University center for clinical research. SUBJECTS: Fourteen nonsmoking, healthy volunteers. INTERVENTION: Subjects received lamotrigine 25 mg/day for 5 days, then 50 mg/day for 10 days to achieve steady-state concentrations. On day 15, blood samples were obtained before and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours after the dose. Lamotrigine 50 mg/day was then given for an additional 3 days. On the next day, lamotrigine 50 mg and olanzapine 5 mg were coadministered. Blood samples were obtained at the same times as before and at 48, 72, and 96 hours after dosing. MEASUREMENTS AND MAIN RESULTS: Blood samples were assayed for lamotrigine and olanzapine concentrations by means of high-performance liquid chromatography. Olanzapine did not significantly affect lamotrigine disposition, as we observed no differences in the area under the concentration-time curve from 0-24 hours or in lamotrigine plasma concentrations at baseline or at 24 hours. For lamotrigine, the mean time to reach maximum concentration was significantly prolonged during olanzapine coadministration (mean +/- SD 1.9 +/- 1.3 vs 4.0 +/- 3.0 hrs, p = 0.025), possibly because of the anticholinergic properties associated with olanzapine. Mild sedation was the only adverse effect that occurred during lamotrigine and olanzapine coadministration. CONCLUSION: Lamotrigine and olanzapine can safely be combined in healthy volunteers at the low doses studied, without a clinically significant interaction. When prescribing high doses of olanzapine and lamotrigine for bipolar disorder, patients must be carefully monitored.

Influence of pH, buffer species, and storage temperature on physicochemical stability of a humanized monoclonal antibody LA298.

The purpose of this work is to study the effect of pH, buffer species, and temperature on the physicochemical stability of a humanized monoclonal antibody LA298. The study was carried out in solution state of the antibody in the presence of different buffer species at different pH values and storage temperature. No significant changes in total protein content were observed for any of the solutions with different buffers at different pH values when stored for 8 weeks at both 5 degrees C and 25 degrees C or at 37 degrees C for 1 week. Known asparagines (Asn55) deamidation of LA298 was found to be dependent on pH, buffer type, and temperature. The estimated rate constant of the double heavy chain Asn55 deamidation in phosphate buffer at pH 6.5 and 7.0 was much higher than that in citrate buffer under the same storage conditions. However, comparable results were obtained for single heavy chain Asn55 deamidation in citrate and phosphate buffer. Aggregation of LA298 was not significant for samples at different pH values, buffers, and temperatures as the monomer of LA298 decreased dramatically over time. Less decrease in monomeric LA298 was observed in citrate buffer, pH 5.0-5.5. In conclusion, to minimize deamidation and loss of LA298 monomer, it is important to optimize its solution pH, buffer species, and storage temperature.

Decrease of genital organ weights and plasma testosterone levels in rats following oral administration of leuprolide microemulsion.

Studies were conducted to develop oral leuprolide microemulsions using oleic acid as an absorption enhancer and to evaluate its absorption and pharmacological responses in rats. Oral administration of leuprolide microemulsion at a dose of 3 mg/kg showed a greater in vivo exposure level (C(max) and AUC) than its saline solution. When male rats were orally given a microemulsion formulation of leuprolide acetate at 0.25, 0.5, and 1mg/day for 14 consecutive days, a significant decrease in testis, prostate and seminal vesicle weights was observed. In a 35-day study, the reduction of the male genital organ weights by once a day treatment (2 mg/rat, qd) was similar to that by twice a day treatment (1 mg/rat, bid) at the same dose level. From both 14- and 35-day studies, plasma testosterone levels were sharply increased at the beginning of the treatment, and then significantly decreased to below normal control level which was also maintained during the treatment. In female rats, similar reduction of uterus and ovary weights was obtained following oral administration of leuprolide microemulsion for 35 days. These antagonistic activities from oral leuprolide microemulsion were similar to a single subcutaneous injection of Lupron depot (3.75 mg/rat), a commercial leuprolide product. The results indicated that leuprolide absorbed into systemic blood circulation from the oral microemulsion containing oleic acid reached the plasma level which can exert its pharmacological effects. Increasing oral absorption of leuprolide observed in this study could be mediated by improved membrane permeation from oleic acid and reduced enzymatic degradation from microemulsions. These findings suggest that systemic absorption of highly water-soluble protein or peptide drugs could be enhanced by oral microemulsions containing oleic acid.

Taste masking analysis in pharmaceutical formulation development using an electronic tongue.

The purpose of this study is to assess the feasibility for taste masking and comparison of taste intensity during formulation development using a multichannel taste sensor system (e-Tongue). Seven taste sensors used in the e-Tongue were cross-selective for five basic tastes while having different sensitivity or responsibility for different tastes. Each of the individual sensors concurrently contributes to the detection of most substances in a complicated sample through the different electronic output. Taste-masking efficiency was evaluated using quinine as a bitter model compound and a sweetener, acesulfame K, as a bitterness inhibitor. In a 0.2 mM quinine solution, the group distance obtained from e-Tongue analysis was reduced with increasing concentration of acesulfame K. This result suggests that the sensors could detect the inhibition of bitterness by a sweetener and could be used for optimization of the sweetener level in a liquid formulation. In addition, the bitterness inhibition of quinine by using other known taste-masking excipients including sodium acetate, NaCl, Prosweet flavor, and Debittering powder or soft drinks could be detected by the e-Tongue. These results further suggest that the e-Tongue should be useful in a taste-masking evaluation study on selecting appropriate taste-masking excipients for a solution formulation or a reconstitution vehicle for a drug-in-bottle formulation. In another study, the intensity of the taste for several drug substances known to be bitter was compared using the e-Tongue. It was found that the group distance was 695 for prednisolone and 686 for quinine, which is much higher than that of caffeine (102). These results indicate that the taste of prednisolone and quinine is stronger or more bitter than that of caffeine as expected. Based on the group distance, the relative intensity of bitterness for these compounds could be ranked in the following order: ranitidine HCl>prednisolone Na>quinine HCl approximately phenylthiourea>paracetamol>>sucrose octaacetate>caffeine. In conclusion, the multichannel taste sensor or e-Tongue may be a useful tool to evaluate taste-masking efficiency for solution formulations and to compare bitterness intensity of formulations and drug substances during pharmaceutical product development.

False carina: a distinct variant of tracheal bronchus.

Tracheal-bronchus is an aberrant bronchus arising from the lateral tracheal wall, superior to the carina. A "False-carina" can be classified as a sub-type. This clinical entity will be defined and the clinical presentation, diagnosis and management of five patients with variations of the anatomical definition of a False-carina, identified at our institution, will be reviewed. Congenital bronchial abnormalities, including False-carina, have important implications in the overall management of the airway. Management can range from expectant in asymptomatic patients to surgical intervention in cases of recurrent respiratory infections. Awareness and understanding of this clinical entity allows for timely investigation, diagnosis and appropriate intervention.

Brush-type chiral stationary phase for enantioseparation of acidic compounds. Optimization of chiral capillary electrochromatographic parameters.

The capillary electrochromatographic separations of three acidic enantiomers (carprofen, coumachlor and warfarin) were studied on a capillary column packed with 5 microm (3R,4S)-Whelk-O 1 chiral stationary phase. The influence of several experimental parameters (mobile phase pH, type of background electrolyte, acetonitrile ratio, temperature, applied voltage and ionic strength) on electroosmotic flow velocity, retention factor, selectivity factor, efficiency, resolution and effectiveness of chiral separation was evaluated. It was notable that the optimum resolution of the acidic enantiomers was achieved at pH 3.0 phosphate buffer, suggesting that capillary electrochromatography in the ion-suppressed mode can be applied for chiral separations of a range of acidic compounds.

Capillary electrochromatography of methylated benzo[a]pyrene isomers. I. Effect of mobile phase tuning.

It is of increasing importance that chromatographic methods be developed for the separation and identification of biological and environmentally harmful compounds such as methylated polycyclic aromatic hydrocarbons (PAH). Capillary electrochromatography (CEC) is fast becoming a useful technique for analysis of PAH, as it offers both high efficiency and superior resolution. The separation of 12 methylated benzo[a]pyrene (MBAP) isomers is a challenge due to the extreme hydrophobicity and structural similarity of these compounds. In this work, we present Part I of our ongoing study, a method for the systematic mobile phase tuning for CEC separation of the 12 MBAP isomers. The CEC experiments were conducted utilizing a CEC-octadecylsilica (ODS) stationary phase and fused-silica capillary [(75 microm I.D., 363 microm O.D.) 36.5 cm total length, 25.0 cm effective length] which was slurry pressure packed in our laboratory. Several mobile phase parameters were manipulated to provide optimum separation. These included acetonitrile (ACN) concentration, tris(hydroxymethyl) aminomethane (Tris) concentration, pH, and addition of a tertiary buffer constituent such as tetrahydrofuran (THF) and isopropanol (IPA) to ACN-aqueous buffer mixtures. Optimum CEC separation conditions were achieved using 75% (v/v) ACN-25% (v/v) 12.5 mM Tris, pH 8.0, and 30 kV at 25 degrees C. These mobile phase conditions were then utilized for Part II of our study, the CEC stationary phase optimization for the separation of 12 MBAP isomers.

Simultaneous enantioseparation and sensitive detection of eight beta-blockers using capillary electrochromatography-electrospray ionization-mass spectrometry.

The feasibility of using vancomycin chiral stationary phase (CSP) and polar organic eluent is investigated for simultaneous enantioseparation of eight beta-blockers using CEC coupled to ESI mass spectrometric detection (ESI-MS). The internally tapered capillaries were utilized to pack CEC-MS columns. As compared to externally tapered columns, the use of internally tapered columns demonstrated enhanced stability, durability, and reproducibility. A mixture containing methanol/ACN/acetic acid/triethylamine at 70:30:1.6:0.2 v/v/v/v was considered as optimum mobile phase since it provided a good compromise between resolution and analysis time. As expected, sheath liquid and ESI-MS parameters mainly influenced the detection sensitivity. Interestingly, structural information of beta-blockers was available by varying the MS fragmentor voltage using in-house CID in the scan mode. In order to maximize the chiral/achiral resolution, various column-coupling approaches using teicoplanin as complementary CSP to vancomycin were tested. Several changes in the elution order of beta-blockers were observed using multimodal CSPs with some improvement in chiral or achiral resolution. The quantitative aspects of the CEC-MS method were demonstrated using R- and S-talinolol as internal standards. The calibration curves of beta-blockers showed good linearity in the range of 3-600 microM. The enantiomer of beta-blockers at a concentration of 30 nM was detectable. Furthermore, both 0.1 and 1% of the S-enantiomer could be precisely quantified in the presence of 99.9 and 99% of the R-isomer of beta-blocker.

Fabrication of internally tapered capillaries for capillary electrochromatography electrospray ionization mass spectrometry.

In this study, we report a novel procedure for fabricating internally tapered capillary columns suitable for the coupling of capillary electrochromatography (CEC) to electrospray mass spectrometry (ESI-MS). The internal tapers were prepared by slowly heating the capillary end in a methane/O2 flame. Due to continuous self-shrinking of the inner channel of the capillary, the inside diameter of the opening was reduced to 7-10 microm. The procedure is easy to handle, with no requirement for expensive equipment as well as elimination of problematic grinding of the tip. Several advantages of these new internal tapers, as compared to using externally tapered columns, are described. First, the problems of poor durability and tip breakage associated with external tapering were successfully overcome with the internal taper. A comparison of the online CEC/ESI-MS between external versus internal tapers showed that the latter provides enhanced electrospray stability, resulting in significantly lower short-term noise and very short-term noise values. In turn, the more rugged design of internal tapers allows performing CEC/MS utilizing a harsh polar organic mobile phase, which was not previously successful using an external taper due to higher operating current and electrospray arcing. Next, data on the reproducibility of the internally tapered CEC/MS column using warfarin and beta-blockers as model analytes are presented. For example, when comparing the reproducibility for separation of warfarin under reversed-phase conditions, the internal taper demonstrated superior intraday % RSD (1.6-3.4) as compared to the external taper intraday % RSD (5-6). Last, the applicability of performing quantitative CEC/MS with internally tapered capillaries is demonstrated for simultaneous enantioseparation of beta-blockers. Impressive quantitative results include good linearity of calibration curves (e.g., R2 = 0.9940-0.9988) and limit of detection as low as 30 nM. The sensitive detection of a minor impurity of one enantiomer at the 0.1% level in a major chiral entity buttresses the suitability of compliance with FDA guidelines.

Application of polymeric surfactants in micellar electrokinetic chromatography-electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs.

Chiral micellar EKC (CMEKC) coupled to ESI-MS using polymeric surfactants as pseudostationary phases is investigated for simultaneous enantioseparation of two benzodiazepines, (+/-)-oxazepam ((+/-)-OXA) and (+/-)-lorazepam ((+/-)-LOR), and one benzoxazocine, (+/-)-nefopam ((+/-)-NEF). First, enantioselectivity and electrospray sensitivity of six chiral polymeric surfactants for all three chiral compounds are compared. Second, using poly(sodium N-undecenoyl-L-leucinate) as pseudostationary phase, the organic modifiers (methanol (MeOH), isopropanol, and ACN) are added into the running buffer to further improve chiral resolution (RS). Next, a CMEKC-ESI-MS method for the simultaneous enantioseparation of two benzodiazepines is further developed by using a dipeptide polymeric surfactant, poly(sodium N-undecenoxy carbonyl-L,L-leucyl-valinate) (poly-L,L-SUCLV). The CMEKC conditions including nebulizer pressure, capillary length, ammonium acetate concentration, pH, poly-L,L-SUCLV concentration, and capillary temperature were optimized to achieve maximum chiral RS and highest sensitivity of MS detection. The spray chamber parameters (drying gas temperature and drying gas flow rate) as well as sheath liquid conditions (MeOH content, pH, flow rate, and ionic strength) were found to significantly influence MS S/N of both (+/-)-OXA and (+/-)-LOR. Finally, a comparative study between simultaneous UV and MS detection showed high plate numbers, better chiral RS, and enhanced detectability with CMEKC-MS. However, speed of analysis was faster using CMEKC-UV.