High Performance Liquid Chromatograpy Research Articles

Novel developed HPLC analyses of [68 Ga]Ga/[177 Lu]Lu-EDTMP and [68 Ga]Ga/[177 Lu]Lu-DOTA-Zoledronate.

Radiolabeled phosphonates have found widespread applications in nuclear medicine for the treatment of bone metastases. Even though considerable attention has been devoted to the administration of radiolabeled phosphonates to the patients, quality control (QC) studies belong to detection of them are still speculative and need to be improved. Herein, practical, efficient, and more reliable high-performance liquid chromatograpy (HPLC) methods were described for the first time. Radiochemical purity was easily determined by our developed analytical methods. N,N-Dimethylhexylamine and N,N-dimethyltetradecylamine were furnished as suitable ion pair reagents for the identification of radiopharmaceutical products. Effects of column type, pH of the mobile phase, and salt concentration were also examined.

Follicular Fluid Zinc Level and Oocyte Maturity and Embryo Quality in Women with Polycystic Ovary Syndrome.

Background:Polycystic ovary syndrome (PCOS) is considered to be one of the most common endocrine disorders in women of reproductive age. Zinc, a vital trace element in the body, plays a key role in maintaining health, especially due to its antioxidant role. On the other hand, lack of antioxidants and oxidative stress can adversely affect oocytes quality and consequently fertility rate. The available studies that report the effect of follicular fluid (FF) zinc in terms of the number and quality of the oocytes in infertile women with PCOS, are few and not consistent. We decided to investigate this issue.Materials and Methods:In this cross-sectional study, from the women with PCOS referring to Omolbanin Hospital, Dezful, Iran (February to December 2019), a total of 90 samples (follicular fluid, oocytes, and embryos) were collected from those who had undergone in vitro fertilization (IVF). To measure zinc level in follicular fluid, high performance liquid chromatograpy (HPLC) was utilized. Also, oocytes maturity and embryos quality evaluation was performed using inverted optical microscopy. One-way ANOVA and Fisher’s least significant difference (LSD) were used for data analysis.Results: The amount of FF zinc was not associated with any significant differences in the number of oocytes and metaphase I (MI) and germinal vesicle (GV) oocytes, but a significant decrease was observed in the number of metaphase II (MII) oocytes at zinc values less than 35 µg/dL. The FF zinc levels less than 35 µg/dL were also significantly associated with decreased embryo qualityConclusion:A significant relationship was found between the level of FF zinc and the quality and the number of oocytes taken from the ovaries of infertile patients with PCOS history who were candidates for IVF treatment as well as the number of high quality embryos.

Enantioseparation on Amino Alcohol‐Derived Stationary Phases by Supercritical Fluid Chromatography and High‐Performance Liquid Chromatograpy

Previous studies have shown that amino alcohol‐derived chiral stationary phases can effectively separate some chiral samples. With the aim at improving the chiral separation, in this study, the composition and type of the mobile phase and the particle size of the stationary phase are changed, and the analytical method is carried out in parallel using high‐performance liquid chromatograpy (HPLC) and supercritical fluid chromatography (SFC). By changing the type and composition of the mobile phase and reducing the particle size of the stationary phase, optimal separation and analysis conditions are obtained simultaneously for HPLC and SFC experiments. In particular, for a particle size of 3 μm, the resolution increases for dinitrobenzoyl phenylglycinol and 2,2,2‐trifluoro‐1‐(9‐anthryl)ethanol. Moreover, replacing the organic modifier from methanol to isopropanol allows obtaining better results in the SFC experiments of dinitrobenzoyl phenylglycinol, dinitrobenzoyl leucinol, and 2,2,2‐trifluoro‐1‐(9‐anthryl)ethanol GTOC,Chiral separation by SFC and HPLC.

Simultaneous determination of macrolide pesticides in fruits and vegetables by liquid chromatography

A multiresidue method was developed and validated for the determination of four macrolide pesticides (spinosad, emamectin benzoate, abamectin and milbemectin) and their metabolites in six kinds of vegetables and fruits (Chinese kale, cabbage, eggplant, orange, papaya and strawberry). The pesticide residues were extracted from the samples with acetonitrile/10 mM aqueous ammonium acetate (NH4OAC), pH 4.0 (90:10, v/v) and the purification was carried out with an aminopropyl (NH2) solid-phase extraction cartridge. Reversed-phase high-performance liquid chromatograpy (RP-HPLC) using an ODS-AM column 5 μm, where a gradient elution with methanol/acetonitrile/10 mM aqueous NH4OAc, pH 4.0 mobile phase was employed for the separation and quantification of the pesticides. The method accuracy and precision were determined via recovery experiments and four replicates spiked with three fortification levels (0.15, 1.5, 7.5 μg/g) each. From the overall results of analytes, method LOD values were ranged from 0.01 to 0.03 μg/g, the mean recoveries were in the range of 75-117% and the relative standard deviations (RSD) were lower than 14%. The recovery data from six sample matrices met the acceptability criteria (European Union, SANCO/2007/3131) of recovery (70-120%) and RSD (<20%) at the three levels studied. The simple and efficient method with acceptable performance was thus established for the simultaneous determination of four macrolide pesticides and their metabolites in agricultural products.

Allelopathic potential of leaf aqueous extracts from Cynara cardunculus L. on the seedling growth of two cosmopolitan weed species

The search for sustainable alternatives to synthetic herbicides for weed control, has led the scientific community to an increased interest for plant allelopathic mechanisms. The utilisation of plant extracts as possible bioherbicides represents an important solution. In the present study, laboratory experiments were carried out to investigate, for the first time, the differences in the allelopathic activity of the three Cynara cardunculus L. botanical varieties (globe artichoke, cultivated and wild cardoon) leaf aqueous extracts on the seedling growth of Amaranthus retroflexus L. and Portulaca oleracea L. In addition, the autoallelopathic effect on wild cardoon growth and the qualitative profile of the aqueous extract through high-performance liquid chromatograpy (HPLC) analysis were evaluated. Overall, the allelopathic effects were both genotype- and weed species-dependent. Wild cardoon showed the highest allelopathic potential (–23.4%), followed by cultivated cardoon and globe artichoke, and P. oleracea was the most sensitive target species (–32%). Besides, root system length was the most affected parameter (–32.6%). The autoallelopathic effect of wild cardoon extract was also demonstrated on root system length, hypocotyl and epicotyl length and total dry weight. C. cardunculus leaf aqueous extract was characterised by 5 sesquiterpene lactones, 2 caffeoylquinic acids, 6 flavones and 1 lignan. From the HPLC analysis we found that apigenin and luteolin 7-O-glucoronide were detected only in wild cardoon, apigenin 7-O-glucoside was typic of globe artichoke, and 11,13-dihydro-deacylcynaropicrin and 11,13-dihydroxi-8-deoxygrosheimin were characteristics of cultivated cardoon.

PENGUKURAN KADAR PIRIDOKSIN (VITAMIN B6) DALAM DARAH PADA ANAK PENDERITA DEFISIT PERHATIAN DAN GANGGUAN HIPERAKTIVITAS (ADHD)

This research aimed to study Piridoksin (Vitamin B6) status in blood sample of Attention Deficit Hyperactivity Disorder (ADHD) children. Deductive research using the descriptive method through 2 stage, namely : (1) Taken of blood sample, and (2) Analysis of Piridoksin (Vitamin B6) status in blood sample was performed by using High Performance Liquid Chromatograpy (HPLC) method. Data collected and assessed by once in one time. Data were analyzed using descriptive analysis techniques and Chi Square tests (X&lt;sup&gt;2&lt;/sup&gt;) by Computer programs (SPSS 15). The result of the assessment of Piridoksin (Vitamin B6) status in blood sample of ADHD children that had been was considered statistically highly significant a reduce of the assessment of Piridoksin (Vitamin B6) status in blood sample of ADHD children by 0,422 ng/ml compared with laboratory reference (control) by 0,36-1,8 ng/ml, HA&amp;lt;C** (p&amp;lt;0,001). This was considered a reduction in Piridoksin (Vitamin B6)) concentrations induces of the Hyperactivity children by 95%.

Eplerenone nanoemulsions for treatment of hypertension. Part I: Experimental design for optimization of formulations and physical characterization

Nanoemulsions of eplerenone (EP) were designed with the aim of improving its bioavailability for an effective antihypertensive therapy. Nanoemulsions were prepared by high shear homogenization and ultrasonication methods. Oleyl erucate, Brij® 35, Tween® 80, Tego Care® 450 and Poloxamer® 407 were used as the liquid lipid and surfactants for optimization of formulations by design of experiments approach. Thus, oil, surfactant and water contents of nanoemulsions were defined by determination of their droplet size and droplet size distribution by dynamic light scattering method. Formulations were screened by scanning electron microscopy. Drug payload and release properties of the formulations were investigated. Analytical quantification method of EP was validated by high performance liquid chromatograpy. Nanoemulsions having average droplet size between 150.6 nm and 205.8 nm were gained with homogenous droplet size distribution and high entrapment efficiency (84.47–98.51%). Formulations released 44.37–82.87% of EP within 1 h and drug release was completed up to 5–6 h. Thus, drug release characteristics of formulations optimized in this study might introduce benefits for rapid response and maintenance of treatment in hypertension attacks. As a result, design of experiments approach provided accurate and consistent datum with those obtained from experiments on the bench for optimization of formulations.

Some Characteristic Properties of Chestnut and Rhododendron Honeys in Turkey

Chestnut (Castanea sativa Mill.) and Rhododendron (Rhododendron spp. L.) (mad honey) honeys are produced generally in Black Sea Region in Turkey and both of them are the special honeys because of their organic component content and known their high antioxidant capacity. In the first step of this study we researched the melissopalynological differentiation of the chestnut and rhododendron honeys and then in the second step we determined the chemical compounds and sugar content of the rhododendron, chestnut and mixed chestnut&rhododendron honeys. For this purpose total 18 honey samples were collected from 4 different districts from Black Sea Region of Turkey and melissopalynological analyses were done by microscope. Chemical composition and sugar content (fructose&glucose) were determined by High Performance Liquid Chromatograpy (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS). After melissopalynological anaylses were obtained 10 monofloral chestnut, 2 monofloral rhododendron and 6 mixed chestnut&rhododendron honeys. As a result of sugar analysis with HPLC, F/G rates were found between 1.17 and 1.80. GC-MS chemical substance analyses of honeys revealed alcohols, aldehydes, ketones, aliphatic acids and their esters, carboxylic acids and their esters and flavanonoids.

Proximate, Amino Acid and Fatty Acid Composition of Eremomastax polysperma, Brillantaisia owariensis Leaves and Sorghum vulgare Leaf-Sheath

Proximate, amino acid and fatty acid composition of Eremomastax polysperma, Brillantaisia owariensis and Sorghum vulgare leaf-sheath was studied. The amino acid content was assayed using High performance liquid chromatograpy while the fatty acid content was assayed using gas chromatograpy. The results from this research, showed that the ash and protein content of Eremomastax polysperma (3.91 ± 0.01%; 3.80 ± 0.05%) was significantly higher than Sorghum vulgare (1.74 ± 0.03%; 1.21 ± 0.05%), and B. owariensis (3.39 ± 0.01%; 1.61 ± 0.01%) respectively. The total essential amino acid composition of Eremomastax polysperma (43.18g/100g) was higher than Brillantaisia owariensis (39.41g/100g) and S. vulgare (31.40g/100g), with lysine and cysteine concentration of B. owariensis (9.72; 9.45) been higher than E. polysperma (9.48;6.59g/ 100g) and S. vulgare (4.11 and 7.19g/100g). The total nonessential amino acid composition of Sorghum vulgare leafsheath (59.49g/100g ) was higher than Eremomastax polysperma (53.09g/100g) and Brillantaisia owariensis (53.82g/100g), with glutamate (14.69, 20.08 and 14.46g/ 100g) and aspartate (12.21, 13.22 and 10.64g/100g) having a higher concentration for E. polysperma, S. vulgare and B. owareinsis respectively. For the fatty acid composition, Sorghum vulgare leaf-sheath (17.51%) had a higher oleic acid concentration than Eremomastax polysperma (1.50%) and Brillantaisia owariensis (5.96%). Brillantaisia owariensis had a higher palmitic acid (35.30%) and behenic acid (14.17%) than Eremomastax polysperma (0.00% and 13.13%) and Sorghum vulgare leaf-sheath(25.96% and 12.21%) . Whereas Eremomastax polysperma had a higher myristic, magaric and linolenic acid concentration (45.91%; 20.27% and 10.45%) than Brillantaisia owariensis (37.23%; 0.36% and 0.00%) and Sorghum vulgare leaf-sheath (37.24%; 0.11% and 0.03%) respectively. The results obtained from this research implies that Eremomastax polysperma, Brillantaisia owariensis leaves and Sorghum vulgare leaf-sheath have nutritional benefits.

HUBUNGAN KADAR TARTRAZIN DAN SENG (ZN) DALAM DARAH PADA ANAK PENDERITA DEFISIT PERHATIAN DAN GANGGUAN HIPERAKTIVITAS (ADHD)

This research aims to study Tartrazine and Zinc (Zn) status in blood sample of Attention Deficit Hyperactivity Disorder (ADHD) children. Deductive research using the descriptive method through 3 stage, namely : 1. Taken of blood sample, 2. Analysis of Tartrazine status in blood sample was performed by using High Performance Liquid Chromatograpy (HPLC) method, and 3. Analysis of Zinc (Zn) status in blood sample were measurements by using Atomic Adsorbance Spectrophotometry (AAS) method. Data were analyzed using descriptive analysis techniques and Chi Square tests (X2) by Computer programs (SPSS 15). The result of the assessment of Tartrazine status in blood sample of ADHD children that had been was considered statistically highly significant a increase of the assessment of Tartrazine status in blood sample of ADHD children by 130 mg/ml compared with laboratory reference (control) by 28 mg/ml, HA&gt;C** (p&lt;0,001). The result of the assessment of Zinc (Zn) status in blood sample of ADHD children that had been was showed statistically highly significant a reduce of the assessment of Zinc (Zn) status in blood sample of ADHD children by 0,37 µg/ml compared with laboratory reference (control) by 0,64-1,18 µg/ml, HA&lt;C** (p&lt;0,001). This was considered a reduction and increase in Tartrazine and Zinc (Zn) concentrations induces of the Hyperactivity children by 95%.

Structure, functional and antioxidant properties in Tunisian beef sausage of a novel polysaccharide from Trigonella foenum-graecum seeds

A novel polysaccharide named Fenugreek Water-Soluble Polysaccharide (FWSP), consisting of a gluconic acid polymer, was isolated from fenugreek seeds. It was structurally characterized by Fourier transformed infrared (FT-IR), X-ray diffraction (XRD), High performance liquid chromatograpy (HPLC), and Nuclear magnetic resonance (NMR) spectroscopies. FT-IR and NMR spectra showed the characteristic bands of polysaccharides. According to XRD spectrum, FWSP is a semi-crystalline polymer. Functional properties of FWSP were investigated based on Water Holding Capacity (WHC), Oil Holding Capacity (OHC), emulsification activity, and foaming ability. FWSP was capable of emulsifying several food-grade oils and hydrophobic compounds, especially corn oil and hexane. The effects of FWSP on oxidative processes in beef sausages during refrigerated (4°C) storage was investigated. The results showed significant inhibition (p<0.05) of lipid and myoglobin oxidation and provided evidence that FWSP is a potent and useful antioxidant for maintaining storage stability of beef sausages, and could replace vitamin C currently used as antioxidant in industry process.

Pengaruh Kandungan Komponen Minor dari Minyak Kelapa Sawit (Elaeis guineensis Jacq.) Terhadap Aktivitas Antioksidan pada Proses Pemurnian Karotenoid

Crude palm oil (CPO) contains some minor component which has antioxidant activity such as carotenoid and vitamin E. However, heat and chemical exposure in extraction processing can decrease the antioxidant activity of that compound. This research was conducted to evaluate the antioxidant activity of the palm oil extract from each stages process. CPO wereextracted with non polar solvent (hexane), then processed through transesterification, solvolytic miscellization and saponification. Concentration of carotene extract was determined using UV-Visible spectrophotometry at λ 446 nm, the composition of α- and β-carotene and total vitamin E using high performance liquid chromatograpy, ester and triglyceride contents by gas chromatography. Carotene antioxidant activity of extracts were tested using 2,2’-diphenyl-1-picrylhidrazyl (DPPH) with incubation time 30 min and 60 min. Carotene concentrations increased according to the stage of the extraction process, where the concentration of extract control reached 514 mcg/ml, rised to 599 mcg/ml in extract I, increased to 7,346 mcg/ml in extract II and rised extremely into 229,968 mcg/ml in extract III. All of extracts has content of β-carotene (64.60-68.56%) was higher than α-carotene (31.44-35.40%). The antioxidant activity (IC 50 ) of carotene extracts in extract Iwith incubation time at t-60 was 5.610 mcg/ml, more powerful than control extract, extract II and extract III. However, all of carotene extracts showed very high antioxidant activity which is lower than 50 mcg/ml.

Unraveling the secretome of Termitomyces clypeatus grown on agroresidues as a potential source for bioethanol production

Termitomyces clypeatus MTCC 5091 is an edible mushroom and is prized for its nutritional value as well as for harboring plethora of enzymes essential for carbohydrate degradation. T. clypeatus when grown on agricultural based carbon sources efficiently induced high quantities of lignocellulolytic enzymes in the secretome. Optimization in tamarind kernal powder (TKP) media through response surface methodology enhanced the enzyme yields by several folds. Correlation between extracellular protein productions of the fungus with respect to its specific growth rate established that secreted proteins were produced most efficiently at low specific growth rates. Proteins released in the T. clypeatus secretome were quantified and identified using SDS-PAGE, 2D gel electrophoreses, zymography and matrix-assisted laser desorption mass spectrometry. 36 proteins identified from the protein spots belonged majority to glucosyl hydrolase family, transporters, uncharacterized and hypothetical proteins. The potential synergistic interactions between the cellulases and xylanases in enzyme preparations of T. clypeatus during hydrolysis of steam pretreated bagasse (SPB) showed improved hydrolysis efficiency and enhanced rate of hydrolysis as observed in high performance liquid chromatograpy. The changes in the ultra-structure of SPB after 12h enzymatic hydrolysis were observed by scanning electron microscopy. The hydrolysates obtained produced ∼7.2g/L ethanol after 6h fermentation determined by gas chromatography.

Abstract P206: Structural Analysis of Glycosyl Chain at 14th Amino Acid Of Human Angiotensinogen in Plasma

The Renin angiotensin system (RAS) is a major regulator of body fluid balance and control of blood pressure. The protease enzyme renin secreted from kidneys cleaves speci[[Unable to Display Character: &amp;#64257;]]cally angiotensinogen (Aogen) circulating in the blood to produce angiotensin I (Ang I). Human Aogen is a heterogeneous glycoprotein constitutively secreted by the liver. In addition, human Aogen contains four putative asparagine-linked glycosylation sites (Asn 14, 137, 271, 295) and contains four cysteines (Cys 18, 138, 232, 308), with Cys18 and Cys138 linked by a disulfide bridge. The glycosyl chains and cysteines position are very important for binding of the renin. Big angiotensin-25 (Bang-25) is a consisting of 25 amino acids with glycosyl chain (14th amino asid) and added cysteine (18th amino acid), which we recently isolated from human urine. To compare glycosyl chain of Bang-25 and Aogen, we analyzed of structure glycosyl chain at position 14th amino acid of human Aogen in plasma. To determine of glycosyl chain at position 14th amino acid, we performed lysyl endopeptidase digestion and reduction on human plasma Aogen. Then, Aogen after digest was purified by reverse-phase high-performance liquid chromatograpy (RP-HPLC), and glycosyl chain structure analyzed by the two-/three-dimensional HPLC mapping method. We show that plasma Aogen has three types of glycosyl chain at position 14th amino acid. One glycosyl chain structure is identical to Bang-25 in urine. N-linked glycosylation on 14th amino acid of Aogen plays an important role about renin reaction. In addition, Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleave by renin. These results suggest that the structure of the glycosyl chain at position 14th amino acid of the human Aogen may be involved in the substrate specificity for renin or chymase.

Prevention of Urinary Tract Infections with Vaccinium Products

Cranberries exert a dose-dependent inhibition of the adherence of E. coli fimbriae to uroepithelial cells. This was demonstrated in vitro but also ex vivo in vitro with urine from cranberry consumers. The active principle has not been identified in detail but type-A proanthocyanidins (PAC) play an important role in the mechanism of action. Since the three species, American cranberry (Vaccinium macrocarpon), European cranberry (Vaccinium oxycoccus) and/or lingonberry (Vaccinium vitis-idaea), have different patterns of type-A PACs, results from one species cannot be transferred to the others. It seems likely that most of the studies with monopreparations from V. macrocarpon were underdosed. Whereas photometric PAC quantification may overestimate the true content on co-active compounds, reversed phase high-performance liquid chromatograpy may underestimate them. Recent studies with PAC doses in the upper range (DMAC method) or declared type-A PAC content in the daily dose reveal a dose-dependent trend of clinical effectiveness, however, with a possible ceiling effect. In order to clarify this, future three-arm studies should investigate Vaccinium preparations with higher type-A PAC doses than previously used. We analysed two popular European vitis-idaea products, a mother juice and a proprietary extract. Both preparations may be appropriate to confirm the Vaccinium urinary tract infection-preventive effect beyond doubt.

Quantification of Glutathione in Plasma Samples by HPLC Using 4-Fluoro-7-nitrobenzofurazan as a Fluorescent Labeling Reagent

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (77:23, v/v) at a flow rate of 1.0 mL/min with the column temperature 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 µmol/L to 10.0 µmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.

Augmentation of Clozapine with Aripiprazole in Severe Psychotic Bipolar and Schizoaffective Disorders: A Pilot Study

Aim: To evaluate the efficacy and safety of the augmentation of clozapine with aripiprazole in patients with treatment-resistant schizoaffective and psychotic bipolar disorders in a retrospective manner. Pharmacodynamic and pharmacokinetic interactions between the two drugs were also investigated.Patients: Three men and 4 women (median age 36 and 40 years, respectively) who had mean scores at BPRS and CGI-Severity of 59.1±12.0 and 5.4±0.5, respectively, were treated with clozapine (mean dose 292.9±220.7 mg/day). Patients received an adjunctive treatment with aripiprazole (mean dose 6.8 ± 3.7 mg/day). Clozapine, norclozapine and aripiprazole plasma levels were measured by means of a high performance liquid chromatograpy with UV detection.Results: Total scores at BPRS decreased significantly (from 59.1±12.0 to 51.1±15.6, p=0.007) after aripirazole augmentation. In particular, the factors “thought disorder” (from 10.4±4.4 to 9.0±4.5, p=.047) and “anergia” (from 10.0±2.7 to 8.0±2.4, p=.018) significantly improved. Concomitant administration of aripiprazole and clozapine did not result in an increase in side effects over the period of treatment. Dose-normalized plasma levels of both clozapine and norclozapine and the clozapine/norclozapine metabolic ratio in all patients did not vary as well.Conclusion:The augmentation of clozapine with aripirazole was safe and effective in severe psychotic schizoaffective and bipolar disorders which failed to respond to atypical antipsychotics. A possible pharmacokinetic interaction between clozapine and aripiprazole does not account for the improved clinical benefit obtained after aripiprazole augmentation.

Photoisomerization of All-transRetinal Triggered with Femtosecond Phase-locked Pulse Pairs

We investigate dependence of photo-isomerization compositions on the time interval of a femtosecond phase-locked pulse pair used for excitation. The compositions were analyzed directly by high-performance liquid chromatograpy. Coherent enhancement of the isomerization from all- trans to 13- and 9- cis retinals was observed via the two-photon excited Ag state. We determined the dephasing time of the excited-state wave-packets to be less than 80 fs.

Determination of highly soluble L-Carnitine in biological samples by reverse phase high performance liquid chromatography with fluorescent derivatization

This study was performed in order to validate an effective high performance liquid chromatograpy (HPLC) method to determine L-carnitine in biological samples such as plasma, milk and muscle in cows. An L-carnitine derivative for fluorescence absorption was synthesized with 1-aminoanthracene (16 mg/mL in acetone) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC; 160 mg/mL in 0.01 M NaH2PO4 buffer) as a precolumn fluorescent derivative reagent. gamma-Butyrobetaine HCI was used as an internal standard. A reversed-phase column with fluorescence detection at the excitation and emission wavelengths of 248 and 418 nm were used. The mobile phase consisted of 30% acetonitrile with 0.1 M ammonium acetate in water (pH 3.5) adjusted with acetic acid and delivered at a flow rate of 1.5 mL/ min. The L-carnitine concentration in plasma, milk and muscle samples of cows after oral feeding with 24 g L-carnitine/day for 2 months was then determined. All biological samples were deproteinated by barium hydroxide and zinc sulfate heptahydrate before the derivative reaction. Blank cow plasma was dialyzed using cellulose membrane for standard calibration. The calibration curve showed good linearity (r2 > 0.999) over the concentration range of 50 to 5000 ng/mL. The precision and accuracy were also satisfactory with less than 15% intra- and interday coefficiency of variations. The peaks of L-carnitine and internal standard in HPLC chromatography were successfully separated in plasma, milk and muscle samples of cows. The current derivatization method of L-carnitine for fluorescence detection was simple and adequately sensitive and could be applied to determine L-carnitine in biological samples.

Abnormal Response to Physical Activity in Femurs after Heterozygous Inactivation of One Allele of the Col2a1 Gene for Type II Collagen in Mice

The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6-TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC). The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and maturation.