Ultra Pressure Liquid Chromatography Research Articles

Impaired sperm function in infertile men relies on the membrane sterol pattern.

Membrane cholesterol removal appears a key step for the gain of fertility potential during sperm maturation. However, the membrane sterol pattern in sperm cells from infertile patients, with impaired sperm parameters, has been poorly investigated. To elucidate a causative link between sperm membrane composition in male fertility, here we have investigated the levels of cholesterol and its oxidized derivatives 7β-hydroxycholesterol and 7-keto-cholesterol in sixteen infertile patients with oligo-asthenozoospermia and 16 normozoospermic (N) fertile subjects. Furthermore, ten of 16N fertile subjects agreed to receive a defined testicular thermal challenge by adhering to a programme of sauna sessions for 1month. Semen samples were obtained from each of the participants, and sperm parameters were assessed according to the World Health Organization criteria. Sperm levels of cholesterol, 7β-hydroxycholesterol and 7-keto-cholesterol were quantified by ultra-pressure liquid chromatography mass spectrometry. The results showed that oligo-asthenozoospermia patients had a huge amount of cholesterol content compared with fertile subjects (12.40±6.05μg/106 cells vs. 0.45±0.28μg/106 cells, p<0.001, N and oligo-asthenozoospermia, respectively). Also, oxidized derivatives were significantly higher in oligo-asthenozoospermia patients (7β-hydroxycholesterol: 1.96±1.03ng/106 cells vs. 0.075±0.05ng/106 cells, p<0.001 and 7-keto-cholesterol: 1.11±0.72ng/106 cells vs. 0.005±0.003ng/106 cells, p<0.001). Moreover, sauna exposure, in parallel with a progressive worsening of sperm motility parameters, was associated with a reversible increase in sperm cholesterol after the third and fourth week of treatment, whilst 7β-hydroxycholesterol and 7-keto-cholesterol levels showed an earlier enhancement starting from the second week. Our data show for the first time in humans a strong difference in the cholesterol and its oxidized derivatives of infertile and fertile subjects. These findings suggest a strict biochemical link relating testis function, sperm membrane status and male fertility potential.

Stability studies of endocrine disrupting tributyltin and triphenyltin compounds in an artificial sea water model.

Triorganotins belong to toxic components present predominantly in antifouling paints for marine vessels. Tributyltin/triphenyltin at pico- or nanomolar concentrations in sea water are known to induce an irreversible sexual abnormality in females of over 190 marine species, an "imposex" phenomenon - the superimposition of male genitalia on a female. Moreover, trialkyltins and triaryltins function as potent nuclear retinoid X receptors (RXR) agonists. In mammals, triorganotin compounds induce immunosuppressive, metabolic, reproductive or developmental effects. Toxic effects of triorganotins warrant the need for monitoring of their long-lasting presence in the environment. This study brings novel data on the stability of two triorganotin compounds in artificial sea water model obtained by applying ultra-pressure liquid chromatography (UPLC) and gas chromatography-mass spectrometry (GC-MS) methods. Stability of tributyltin and triphenyltin chlorides was studied for 180 days and the degradation kinetic parameters were obtained. Tributyltin chloride was the less stable with the degradation kinetic parameters Kdeg = 0.00014 day-1 and t1/2 = 4950 days (13.6 years). Kdeg of the more stable triphenyltin chloride was determined to be Kdeg = 0.00006 day-1 with t1/2 = 11550 days (31.6 years). Since similar stability data of triorganotin compounds were not published previously, we report high stability for both tested compounds, which indicates a significant environmental problem when these substances enter sea water and later coastal sediments.

Unexpected Solubility Enhancement of Drug Bases in the Presence of a Dimethylaminoethyl Methacrylate Copolymer.

The methacrylate copolymer Eudragit EPO (EPO) has previously shown to greatly enhance solubilization of acidic drugs via ionic interactions and by multiple hydrophobic contacts with polymeric side chains. The latter type of interaction could also play a role for solubilization of other compounds than acids. The aim of this study was therefore to investigate the solubility of six poorly soluble bases in presence and absence of EPO by quantitative ultrapressure liquid chromatography with concomitant X-ray powder diffraction analysis of the solid state. For a better mechanistic understanding, spectra and diffusion data were obtained by 1H nuclear magnetic resonance (NMR) spectroscopy. Unexpected high solubility enhancement (up to 360-fold) was evidenced in the presence of EPO despite the fact that bases and polymer were both carrying positive charges. This exceptional and unexpected solubilization was not due to a change in the crystalline solid state. NMR spectra and measured diffusion coefficients indicated both strong drug-polymer interactions in the bulk solution, and diffusion data suggested conformational changes of the polymer in solution. Such conformational changes may have increased the accessibility and extent of hydrophobic contacts thereby leading to increased overall molecular interactions. These initially surprising solubilization results demonstrate that excipient selection should not be based solely on simple considerations of, for example, opposite charges of drug and excipient, but it requires a more refined molecular view. Different solution NMR techniques are especially promising tools to gain such mechanistic insights.

Determination of olopatadine in human tears by hydrophilic interaction liquid chromatography-MS/MS method.

The objective of the study was development of hydrophilic interaction liquid chromatography-ESI/MS/MS method for the determination of olopatadine in tear matrix. Separation was performed on Acquity BEH amide column (2.1 × 100mm, 1.7μm). The mobile phase was consisted of 0.1% formic acid in water and acetonitrile. Mianserin hydrochloride was implemented as an internal standard. The artificial tear fluid was used as matrix. The tear samples were collected using Schirmer test strips. For the optimization of ultra pressure liquid chromatography conditions, Box-Benhken design was utilized. The optimal values of the ion source and collision cell parameters were found. Quantification was performed in multiple reaction monitoring mode. The optimized method was fully validated. The proposed method was utilized for monitoring of olopatadine in human tear.

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S.cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MSE). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1.

Effective bioremoval of syntan using fungal laccase to reduce pollution from effluent

Syntans are synthetically prepared tanning agents from phenols that play an important role in leather making by enhancing the filling, grain characteristics and dyeing property. A considerable amount of syntan emanated in waste water contributes high pollution load with other heterocyclic aromatic compounds and causes serious environmental impact resulting in high oxygen demand. In order to overcome this, suitable biodegradation techniques have been developed using laccase from Penicillium chrysogenum. The influence of different environmental parameters on the biodegradation has also been studied which reveals that the maximum syntan degradation was obtained at pH 5.0, temperature at 32 °C for the duration of 48 h. Maximum biodegradation was found to be 96 and 94% for samples containing phenolic and melamine syntan. Ultraviolet spectra showed the peaks at around 280 nm for the presence of phenolic (DI) syntan and at 220 nm for melamine syntan which were disappeared later in the experimental sample indicating complete degradation of syntans. Fourier spectral analysis indicated that the peaks at the region between 1443–1574 and 1176 cm−1 correspond to C=C stretching and C–H bending for aromatic region which were later disappeared in the experimental sample. Ultrapressure liquid chromatography elution profiles of syntans showed relatively shorter retention time indicating formation of oxidized products. Syntans, namely DI and FB6, showed reduction in chemical oxygen demand up to 87.71 ± 4 and 83.38 ± 5%, respectively, while total organic carbon reduction was achieved up to 82.37 and 80.72%, respectively. Toxicity studies revealed that seeds were well germinated using the treated (biodegradation) samples by crude laccase .

Heterologous production of clavulanic acid intermediates in Streptomyces venezuelae

Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS), the production of following clavulanic acid intermediates in S. venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. venezuelae YJ028.

Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography-Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-μm Particles.

In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 μm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Rootstock effects on polyphenol content in grapes of ‘Regent’ cultivated under cool climate condition

Rootstocks are well known as the most efficient way to limit phylloxera. However, they can be useful in order to improve grape quality. This study aimed to compare the content of polyphenol compounds in vine fruits of the cultivar ‘Regent’ grafted on ‘Couderc 161-49’, ‘Sori’, ‘Kober 125AA’, ‘Borner’ or ‘Kober 5BB’ rootstocks, or planted on own-roots. Grape samples were collected in three consecutive seasons (2013-2015) at a research station of the West Pomeranian University of Technology Szczecin in Poland. Thirty-three phenolic compounds were determined in the juice of examined samples using ultra-pressure liquid chromatography with photodiode array and mass spectrometry (UPLC-PDA/MS) method. A significant influence of rootstock on the content of polyphenols in grapes has been proven. The highest content of polyphenols was shown in fruits from a scion grafted on ‘Sori’ and ‘Kober 125AA’ rootstocks (675 and 643 mg · 100 g-1 FW, respectively). ‘Borner’ and ‘Kober 5BB’ rootstocks did not have a significant influence on the creation of polyphenol compounds in comparison to own-root plants. In addition, the use of the ‘Borner’ rootstock resulted in fruits with an especially low content of phenolic acids.

Advanced Oxidation Processes based on the use of UVC and simulated solar radiation to remove the antibiotic tinidazole from water

The objective of this study was to analyse the effectiveness of different Advanced Oxidation Processes based on solar and ultraviolet C radiation to remove the antibiotic tinidazole (TNZ) from water. Specifically the systems studied were: UVC, solar radiation, UVC/H2O2, solar/H2O2, UVC/K2S2O8, solar/K2S2O8, UVC/photo-Fenton and solar/photo-Fenton. Some aspects of these processes such as the degradation percentage of TNZ, its mineralisation degree, and the degradation products, were analysed. The direct photodegradation of TNZ showed low yields, with a removal percentage lower than 50%. With the UVC/H2O2 system, the percentage of TNZ degraded increased with the H2O2 concentration, but when solar radiation was used, the addition of H2O2 did not favour the process efficiency. The UVC/K2S2O8 system was more efficient than UVC/H2O2 for TNZ degradation. The presence of K2S2O8 in the solar photodegradation also enhanced TNZ degradation in comparison to the direct photolysis process. In the photo-Fenton systems, for both UVC and solar radiations, high levels of TNZ degradation and mineralisation were obtained. Therefore, the photo-Fenton systems are the most recommended to treat water contaminated with TNZ. Some clues of the mineralisation process appear from the degradation products detected by means of coupled ultra-pressure liquid chromatography and high-resolution mass spectrometry.

UPLC Method Development and Validation for the Determination of Chlophedianol Hydrochloride in Syrup Dosage Form

A specific, precise, accurate ultra pressure liquid chromatography (UPLC) method is developed for estimation of chlophedianol hydrochloride in bulk drug and syrup dosage form. The method employed with Hypersil BDS C18 (100 mm x 2.1 mm, 1.7 μm) in a gradient mode, with mobile phase of methanol and acetonitrile in the ratio of 65:35 %v/v. The flow rate was 0.1 ml/min and effluent was monitored at 254 nm. Retention time was found to be 1.130±0.005 min. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ)in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was good linear relationship between response and concentration in the range of 20-100 μg/ml respectively. The LOD and LOQ values were found to be 2.094(μg/ml)and 6.3466(μg/ml)respectively. No chromatographic interference from syrup excipients and degradants were found. The proposed method was successfully used for estimation of chlophedianol hydrochloride in syrup dosage form.

Assessment of Biological Activity and Comparison of UPLC and RP-HPLC Chromatographic Profiles of Clausena excavata Burm.F.

Clausena excavata Burm. F. (Rutaceae) has long been associated with medicinal benefits in folk medicine, particularly in the treatment of cancer and its related disorders in the eastern region of Thailand. In the the present study Ultra Pressure Liquid Chromatography (UPLC) analysis of methanolic leaf extracts of Clausena excavata were carried out using short (5 cm × 2.1 mm I.D.) columns, containing 2 μm particles at very high pressures (up to 16000 psi) to obtain high efficiency separations in very short run times. The results indicate that UPLC provided better resolution compared to routine RP-HPLC. The analysis time was drastically reduced (4 min.) which is proved to be a faster and more economical analytical tool than RP-HPLC. In addition to that, the present study also evaluates anti-fungal activity against dermatophytes Trichophyton rubrum and Trichophyton mentographytes by using an agar incorporation technique. The anti-fungal activity may be due to the presence of Angelecin and gallic acid in the plant extract.

On-line solid-phase extraction method for determination of triazine herbicides and degradation products in seawater by ultra-pressure liquid chromatography–tandem mass spectrometry

A fast, simple, selective and sensitive method has been developed for the determination of nine triazine herbicides (ametryn, atrazine, cyanazine, prometryn, propazine, simazine, simetryn, terbuthylazine and terbutryn) and eight degradation products (desethyl atrazine, desethyl-desisopropyl atrazine, desethyl 2-hydroxyatrazine, desethyl terbuthylazine, desisopropyl atrazine, desisopropyl 2-hydroxyatrazine, 2-hydroxyatrazine and 2-hidroxyterbuthylazine) in seawater samples. On-line solid-phase extraction coupled with ultra-pressure liquid chromatography-tandem mass spectrometry was employed for simultaneous analysis of all compounds in 11min. Validation parameters were studied through the estimation of the limits of detection and quantification, calibration curves and precision. Limits of quantification ranged from 0.023 to 0.657μgL−1. Good linearity was obtained for all compounds with R2>0.99 in all cases. Furthermore, inter-day precision (0–2.1%) and intra-day precision (0–3.9%) were shown to be satisfactory. On-line solid-phase extraction recoveries in spiked unpolluted seawater sample were evaluated and acceptable values (80.3–99.8%) with adequate RSD (0.1–3.1%) were found.Finally, the proposed method was applied to the analysis of the target compounds in seawater samples collected from seawater nearby a zone of intensive horticulture of Matosinhos (Portugal). The concentrations of the herbicides were below the limit of detection in all cases.

Simultaneous determination of six active metabolites in Astragalus mongholicus (Fisch.) Bge. under salt stress by ultra-pressure liquid chromatography with tandem mass spectrometry.

Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus, family Leguminosae) is one of the most important traditional Chinese herbs because it contains lots of bioactive metabolites, which have beneficial and pharmacological effects on health. Simultaneously, it has been proved to be a salt-tolerant plant—one of the potential species to control the soil salinization. Therefore, a sensitive and specific ultra-pressure liquid chromatography coupled with tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the simultaneous determination of six main bioactive metabolites, astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin in different organs of A. mongholicus. The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization source operating in the positive ionization mode. Calibration curves offered linear ranges of two orders of magnitude with R2 > 0.99. The method was fully validated for the linearity, intra-day and inter day precisions, accuracy, recovery, matrix effect and stability. Then this method was successfully applied to detect the content of major bioactive metabolites in different plant organs of A. mongholicus under salt stress. Significant variations in the content of six bioactive metabolites were observed after been processed by different levels of salinity in different part of plant. The results support for further exploration of the salt-tolerant mechanisms in A. mongholicus and its possibility as the species that control the soil salinization. Meanwhile, we established a UPLC-MS/MS assay of the trace components in seedling of A. mongholicus in this study.

Ultrapressure liquid chromatography–tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for quantification of 4-methoxydiphenylmethane in pharmacokinetic evaluation

4-Methoxydiphenylmethane (4-MDM), a selective augmenter of Leukotriene A4 Hydrolase (LTA4H), is a new anti-inflammatory compound for potential treatment of chronic obstructive pulmonary disease (COPD). Currently, there is no liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the quantification of 4-MDM. A major barrier for developing the LC–MS/MS method is the inability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) to ionize 4-MDM due to its hydrophobicity and lack of any functional group for ionization. With the advent of atmospheric pressure photoionization (APPI) technique, many hydrophobic compounds have been demonstrated to ionize by charge transfer reactions. In this study, a highly sensitive ultrapressure liquid chromatography tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC–APPI-MS/MS) for the quantifications of 4-MDM in rat plasma has been developed and validated. 4-MDM was extracted from the plasma by solid phase extraction (SPE) and separated chromatographically using a reverse phase C8 column. The photoionization (PI) was achieved by introducing anisole as a dopant to promote the reaction of charge transfer. The assay with a linear range of 5 (LLOQ)–400ngmL−1 met the regulatory requirements for accuracy, precision and stability. The validated assay was employed to quantify the plasma concentrations of 4-MDM after an oral dosing in Sprague Dawley (SD) rats.

Development and validation of a high throughput UPLC–MS/MS method for simultaneous quantification of esomeprazole, rabeprazole and levosulpiride in human plasma

A high throughput ultra pressure liquid chromatography–mass spectrometry (UPLC–MS/MS) method with good sensitivity and selectivity has been developed and validated for simultaneous quantification of esomeprazole, rabeprazole and levosulpiride in human plasma using lansoprazole as internal standard (IS). The extraction method based on liquid–liquid extraction technique was used to extract the analytes and IS from of 50µL of human plasma using methyl tert-butyl ether:ethyl acetate (80:20, v/v), which offers a high recovery. Chromatographic separation of analytes and IS was achieved on a Hypersil gold C18 column using gradient mobile phase consisting of 2mM ammonium formate/acetonitrile. The flow rate was set at 0.5mL/min to elute all the analytes and IS within 1.00min runtime. Detection of target compounds was performed on a triple quadruple mass spectrometer by multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). Method validation results demonstrated that the developed method has good precision and accuracy over the concentration ranges of 0.1–2000ng/mL for each analyte. Stability of compounds was established in a battery of stability studies, i.e., bench top, autosampler, dry extract and long-term storage stability as well as freeze-thaw cycles. The validated method has been successfully applied to analyze human plasma samples for application in pharmacokinetic studies.

Blood plasma lipidome profile of dairy cows during the transition period.

BackgroundThe transition period of dairy cows, around parturition and the onset of lactation, involves endocrine and metabolic changes to compensate for an increased energy requirement aggravated by reduced feed intake. Transition cows adjust to the resulting negative energy balance with the mobilization of lipids from the adipose tissues yielding increased blood levels of non-esterified fatty acids and ketone bodies like β-hydroxybutyrate.ResultsTo study the biochemical adaptations underlying this physiologic adjustment and possible pathologic derangements, we analyzed the blood plasma lipidome of transition cows by ultra-pressure liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry. The resulting data were processed by principal component analysis, revealing over 60 lipid masses that change in abundance over the test period ranging from two weeks before calving to four weeks postpartum. Further characterization of analytes by tandem mass spectrometry demonstrated that the concentration of triacylglycerides in plasma drops at the day of parturition whereas the plasma level of many phosphatidylcholines and two sphingomyelins increases steadily during early lactation.ConclusionThis newly identified shift in phospholipid composition delivers a potential biomarker to detect aberrant metabolic pathways in transition cows and also provides insights into how to prevent and treat associated disorders like fatty liver disease.

Development and validation of an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantitative determination of rhamnolipid congeners.

Rhamnolipids (RLs) are synthesised as a complex mixture of congeners comprising either one or two molecules of rhamnose glycosidically linked to a dimer of 3-hydroxy fatty acids varying in chain length and degree of saturation. Currently, HPLC-MS/MS is the most precise and accurate method for RL determination, while accurate quantification is limited. In this study, a rapid ultra pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the rapid and quantification of individual RL congeners. Increased RLs specificity was achieved using tandem mass spectrometry in multiple reaction monitoring (MRM) mode which was used to quantify RL isomer pairs such as Rha-Rha-C8-C10/Rha-Rha-C10-C8 which are difficult to resolve chromatographically. UPLC showed an 18-fold reduction in retention time for Rha-Rha-C10-C10 (1.07 min) and a 17-fold reduction for Rha-C10-C10 (1.36), the major rhamnolipids present, compared to HPLC, with a total run time less than 2.2 min. The results show that the linear range for the main RL congeners (Rha-C10-C10 and Rha-Rha-C10-C10) is 0.1 to 100 μg/mL. The LOD and LOQ for Rha-C10-C10 is 0.05 and 0.1 μg/mL and for Rha-Rha-C10-C10 is 0.1 and 0.5 μg/mL, respectively. The method was validated for linearity, intra- and inter-day precision and accuracy in accordance with FDA guidelines. The method was applied for the quantification of 14 individual RL congeners produced by Pseudomonas aeruginosa ST5 and comparison of RLs composition on four different carbon sources. Quantification of the individual congeners showed a conserved congener distribution irrespective of carbon source with a preferential selection for C10 β-hydroxyacids as the lipid component of RLs. The only statistically significant differences detected were between actual RL yields on the various carbon sources.

Simultaneous Determination of Multiple Preservatives and Antioxidants in Topical Products by Ultra-Pressure Liquid Chromatography with Photo Diode Array Detector

An improved analytical method was developed by ultra-pressure liquid chromatography (UPLC) using design of experiments (DoE) for quantification of preservatives and antioxidants. Benzyl alcohol, benzoic acid, phenoxy ethanol, sorbic acid, methyl paraben, ethyl paraben, propyl paraben, and butyl paraben were selected as preservatives for the study. Similarly, propyl gallate, butylated hydroxy anisole, butylated hydroxy toluene, and ascorbyl palmitate were selected as antioxidants. Cream, gel, ointment, and lotion formulations containing these components were manufactured and quantified using this method. Separation was performed by liquid chromatography (LC) using reverse phase silica base C18 (2.1 mm × 100 mm, 1.7 µ) column equipped with photo diode array detector. 0.1% v/v ortho-phosphoric acid in water, methanol, and acetonitrile were used for gradient elution. Moreover, extraction and solution stability study of highly oxidizable compound ascorbyl palmitate was performed using a peculiar combination of diluents. The method was found to be simple, precise, accurate, and stability indicating. The applicability of the method was also suggested by evaluating the marketed topical formulations.

Effect of Insulin Sensitizer Therapy on Amino Acids and Their Metabolites

AimsPrior studies have reported that elevated concentrations of several plasma amino acids (AA), particularly branched chain (BCAA) and aromatic AA predict the onset of type 2 diabetes. We sought to test the hypothesis that circulating BCAA, aromatic AA and related AA metabolites decline in response to the use of insulin sensitizing agents in overweight/obese adults with impaired fasting glucose or untreated diabetes. MethodsWe performed a secondary analysis of a randomized, double-blind, placebo, controlled study conducted in twenty five overweight/obese (BMI ~30kg/m2) adults with impaired fasting glucose or untreated diabetes. Participants were randomized to three months of pioglitazone (45mg per day) plus metformin (1000mg twice per day, N=12 participants) or placebo (N=13). We measured insulin sensitivity by the euglycemic-hyperinsulinemic clamp and fasting concentrations of AA and AA metabolites using ultra-pressure liquid chromatography tandem mass spectrometry before and after the three-month intervention. ResultsInsulin sensitizer therapy that significantly enhanced insulin sensitivity reduced 9 out of 33 AA and AA metabolites measured compared to placebo treatment. Moreover, insulin sensitizer therapy significantly reduced three functionally clustered AA and metabolite pairs: i) phenylalanine/tyrosine, ii) citrulline/arginine, and iii) lysine/α-aminoadipic acid. ConclusionsReductions in plasma concentrations of several AA and AA metabolites in response to three months of insulin sensitizer therapy support the concept that reduced insulin sensitivity alters AA and AA metabolites.