Ion-pair high-performance liquid chromatographic assay method for the assessment of clarithromycin stability in aqueous solution and in gastric juice

A simple and selective ion-pair HPLC method has been developed for the analysis of clarithromycin in aqueous solutions and in gastric juice. A Hypersil ODS 5-μm (150 × 4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer (pH 4.6) containing 5 mM 1-octanesulphonic acid (50:50, v/v). The column temperature was 50°C and detection was by UV absorption (210 nm). The limits of detection of 50-μl samples were 0.4 μg/ml (aqueous) and 0.78 μg/ml (0.5 ml gastric juice) or better. The assay was linear in the range of 1.56 to 100 μg/ml with r² values greater than 0.99. The recovery from the gastric juice samples was 98.5±2.9%. The method was applied successfully to determine the stability of clarithromycin in 0.01 M HCl and gastric juice.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4?×?10⁴ to 2.1?×?10⁶?CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100 %), followed by Penicillium chrysogenum (62 %), Aspergillus niger (54 %), and Aspergillus flavus (44 %). Citrinin was present in 100 % samples (0.23–20.65 mg/kg), aflatoxin in 92 % samples (0.61–77.33 μg/kg) and Ochratoxin-A in 100 % samples (0.23–2.48 μg/kg); 100 % citrinin and 76.09 % aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.     

Quantitative high-performance liquid chromatography-based detection method for calphostin C,a naturally occurring perylenequinone with potent antileukemic activity

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca²⁺-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250×4 mm LiChrospher 100, RP-18 (5 μm) in conjunction with a 4×4 mm LiChrospher 100, RP-18 guard column (5 μm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile–water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05–40 μM for calphostin C in 50 μl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 μl plasma was 0.02 μM at a signal-to-noise ratio of ∼3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.     

Optimization of photosynthesis,growth, and biochemical composition of the microalga <Emphasis Type="Italic">Rhodomonas salina</Emphasis>—an established diet for live feed copepods in aquaculture

The cryptophyte Rhodomonas salina is widely used as feed for copepod cultures. However, culturing conditions to obtain high-quality algae have not yet been efficiently optimized. Therefore, we aimed to develop a cultivation protocol for R. salina to optimize its nutritional value and provide technical recommendations for later large-scale production in algal photobioreactors. We studied photosynthesis, growth, pigments, fatty acid (FA) and free amino acid (FAA) composition of R. salina cultured at different irradiances (10–300 μmol photons m^?2 s^?1) and nutrient availability (deficiency and excess). The optimal range of irradiance for photosynthesis and growth was 60–100 μmol photons m^?2 s^?1. The content of chlorophylls a and c decreased with increasing irradiance while phycoerythrin peaked at irradiances of 40–100 μmol photons m^?2 s^?1. The total FA content was maximal at optimal irradiances for growth, especially under nutrient deficiency. However, highly unsaturated fatty acids, desired components for copepods, were higher under nutrient excess. The total FAA content was highest at limiting irradiances (10–40 μmol photons m^?2 s^?1) but a better composition with a higher fraction of essential amino acids was obtained at saturated irradiances (60–140 μmol photons m^?2 s^?1). These results demonstrate that quality and quantity of FA and FAA of R. salina can be optimized by manipulating the irradiance and nutrient conditions. We suggest that R. salina should be cultivated in a range of irradiance 60–100 μmol photons m^?2 s^?1 and nutrient excess to obtain algae with high production and a balanced biochemical composition as feed for copepods.     

Diversity of low-molecular weight organic acids synthesized by <Emphasis Type="Italic">Salix</Emphasis> growing in soils characterized by different Cu,Pb and Zn concentrations

The aim of the study was to evaluate the biosynthesis and exudation of 10 low-molecular weight organic acids (LMWOAs) into the rhizosphere with a simultaneous analysis of the acid contents in the roots and leaves of 9 Salix taxa growing on two experimental areas, differing in their concentrations of copper (Cu), lead (Pb) and zinc (Zn) in the soil (Area 1—low, Area 2—high concentration). The obtained results reveal a significant difference in the phytoextraction of the tested Salix taxa for the analysed metals in both areas. The highest contents of Cu, Pb and Zn were observed for all Salix collected from Area 2, especially in S. × smithiana roots (116 ± 8.76, 87.84 ± 7.30 and 203.42 ± 14.62 mg kg^?1 DW, respectively). The results obtained in Area 2 also revealed acidification of the rhizosphere and a higher concentration of acids, mainly oxalic, malic, malonic, acetic and citric acids. Contents of oxalic, malic, acetic and citric acids increased in the roots of Salix taxa from Area 2, while in the leaves formic and succinic acids were also present. S. × smithiana was the taxon with the highest concentration of acids in the rhizosphere and roots (73.48 ± 6.77 and 49.79 ± 2.65 μM 100 g^?1 DW, respectively), while in leaves a higher content was observed for S. alba and S. viminalis ‘PR’ taxa (78.12 ± 3.95 and 71.12 ± 3.75 μM 100 g^?1 DW, respectively).     

Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-l-cysteine

Polyamines (putrescine, spermine and spermidine) play a crucial role in the regulation of cell growth, differentiation, death and function. Accurate measurement of these substances is essential for studying their metabolism in cells. This protocol describes detailed procedures for sample preparation and HPLC analysis of polyamines and related molecules (e.g., agmatine and cadaverine) in biological samples. The method is optimized for the deproteinization of samples, including biological fluids (e.g., 10 μl), plant and animal tissues (e.g., 50 mg), and isolated/cultured cells (e.g., 1 × 10⁶ cells). The in-line reaction of polyamines with o-phthalaldehyde and N-acetyl-l-cysteine yields fluorescent derivatives which are separated on a reversed-phase C₁₈ column and detected by a fluorometer at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total running time for each sample (including column regeneration on the automated system) is 30 min. The detection limit is 0.5 nmol/ml or 0.1 nmol/mg tissue in biological samples. The assays are linear between 1 and 50 μM for each of the polyamines. The accuracy (the nearness of an experimental value to the true value) and precision (agreement between replicate measurement) of the HPLC method are 2.5–4.2 % and 0.5–1.4 %, respectively, for biological samples, depending on polyamine concentrations and sample type. Our HPLC method is highly sensitive, specific, accurate, easily automated, and capable for the analysis of samples with different characteristics and small volume/amount, and provides a useful research tool for studying the biochemistry, physiology, and pharmacology of polyamines and related substances.     

Fluorometric analysis of N alpha-methylamino acids using fluorescamine

The fluorometric amino acid analyzer based on fluorescamine has been utilized for quantitative determination of N^α-methylamino acids. N-Chlorosuccinimide (1 × 10^?3m in 0.05 m HCl) was continuously introduced into the column eluate to convert N^α-methylamino acids to fluorescamine-sensitive methylamine. As little as 100 pmoles of l-N-methylalanine was detectable with a linear fluorescence response up to 10.0 nmoles. Distinction of primary and secondary amino acids was achieved by carrying out duplicate analyses with and without the introduction of the N-chlorosuccinimide solution.     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

Differential rate of ribosomal protein synthesis in Escherichia coli B-r

The differential rate of ribosomal protein synthesis, α_r (ribosomal protein synthesis rate/total protein synthesis rate), was measured for Escherichia coli strain B/r growing at different steady-state rates ranging from 0.67 to 2.3 doublings/hour. For growth rates above 1.2 doublings/hour, α_r was found to be proportional to the growth rate μ (doublings/h), such that α_r = 0.09 μ, and the ribosome efficiency (amino acids polymerized/second per ribosome), calculated from α_r, was found to be 14 to 18 amino acids/second per ribosome. With decreasing growth rates below 1.2 doublings/hour, α_r was found to be increasingly greater than 0.09 μ and the ribosome efficiency gradually decreased such that at μ = 0.67, α_r = 0.085, and the ribosome efficiency was reduced by 30% and was equal to 10 to 13 ammo acids/second per ribosome. These results imply that the protein to DNA ratio is constant for μ > 1.2 and equal to 4 × 10⁸ to 5 × 10⁸ amino acids/genome. For μ < 1.2, this ratio gradually decreases such that at μ = 0.67, protein to DNA = 3 × 10⁸ to 4 × 10⁸ amino acids/genome. These relationships were verified by direct measurements of the amounts of DNA, RNA and protein at different steady-state growth rates. In addition, protein accumulation was measured following a nutritional shift-up from succinate to glucose minimal medium. The results indicate that the ribosome efficiency increases by approximately 40% within the first few minutes following the shift-up.     

Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins

Reversed-phase HPLC conditions for simultaneous separation of salmon calcitonin, mono- and di-radioiodinated salmon calcitonins and their tryptic digested fragments have been developed. Salmon calcitonin was radioiodinated with Na¹²⁵I by the iodo-beads method. After solid-phase extraction from the reaction mixtures using C₁₈ Bond Elut cartridges, mono- and di-radioiodinated salmon calcitonins were separated from each other, as well as from unlabeled salmon calcitonin, on a Bondclone 10 C₁₈ column (300×7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evaluated on the basis of UV absorbance (215 and 280 nm), fluorescence (λ_ex=282 nm, λ_em=310 nm) and measurement of specific radioactivity by means of a flow-through radio-isotope detector. HPLC separation of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C₁₈ 300 Å column (250×4.6 mm I.D.) and subsequent amino acid analysis, led to the conclusion that radioiodination took place at the Tyr residue and not at the His moiety.     

Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4

A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His₆-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K _m and V _max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min^?1 for l-alanine, and 9.95 mM and 702.6 μmol min^?1 for d-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.     

Highly sensitive determination of TS-962 (HL-004), a novel acyl-CoA:cholesterol acyltransferase inhibitor,in rat and rabbit plasma by liquid chromatography and atmospheric pressure chemical ionization-tandem mass spectrometry combined with a column-switching technique

A quantitative bioanalytical method with excellent specificity using liquid chromatography (LC) atmospheric pressure chemical ionization-tandem mass spectrometry (APCI-MS–MS) combined with a column-switching technique has been developed for the highly sensitive and reliable determination of TS-962 (HL-004), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rat and rabbit plasma. The method involves protein precipitation of a 25-μl aliquot of plasma sample with eight volumes of methanol containing a deuterium-labeled internal standard, the direct injection of a methanolic supernatant into the analytical instrumentation with no sample evaporation and reconstitution steps, automated on-line clean-up on a C₁₈ short trapping column (10 mm×4.0 mm I.D.) followed by separation on a C₁₈ analytical column (50 mm×4.6 mm I.D.), and detection with APCI-MS–MS using m/z 448 ([M+H]⁺) as a precursor ion and m/z 178 as a product ion in a selected reaction monitoring mode. The lower limit of quantification was 1 ng/ml, and good linearity of the calibration graph was obtained in the range of 1∼490 ng/ml with excellent reliability. The developed method enabled pharmacokinetic profiles to be determined for rats and rabbits with sequential plasma collection from an individual animal.     

Omp85 genosensor for detection of human brain bacterial meningitis

The 5′-thiolated DNA probe based on specific virulence gene, Omp85, was immobilized onto a screen-printed gold electrode followed by hybridization with 6–100 ng/6 μl (5.9 × 10⁵–9.3 × 10⁶ c.f.u.) of Neisseria meningitidis single stranded genomic DNA (ssG-DNA) for 10 min at 25 °C from the cerebrospinal fluid (CSF) of a meningitis patient. The Omp85 genosensor can detect as little as 6 ng ssG-DNA in 6 μl CSF of a human brain meningitis patient in 30 min including a response time of 1 min by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance. The sensitivity of the genosensor electrode was 2.6(μA/cm²)/ng using DPV with regression coefficient (R²) 0.954. The genosensor was characterized using Fourier transform infrared spectroscopy and atomic force microscopy. Omp85 genosensor was stable for 12 months at 4 °C with 12 % loss in DPV current.     

Characterization of a novel steviol-producing β-glucosidase from Penicillium decumbens and optimal production of the steviol

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing β-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3–6.0 and at 40–60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and ¹H- and ¹³C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other β-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 μl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel β-glucosidase, which may be useful for the pharmaceutical and agricultural areas.     

Purification and Characterization of a Proteinaceous Toxin from the Serum of Japanese Eel Anguilla japonica

Although eels are well known to contain toxins in the serum, their chemical properties have remained to be clarified for a long time. In this study, a proteinaceous toxin was purified from the serum of Japanese eel Anguilla japonica by anion-exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC. The toxin was lethal to both mice and crabs; the LD₅₀ of the purified toxin against mice (intravenous injection) and crabs (injection into body cavity) were estimated to be 670 and 450 μg kg^?1, respectively. Chemical analysis data revealed that the toxin is a monomeric simple protein with a molecular mass of 100 kDa and an isoelectric point of 6.1. Three of the peptide fragments produced by digestion of the purified toxin with lysylendopeptidase were sequenced. However, a database search based on the determined partial amino acid sequence failed to find any proteins sharing homology with the A. japonica serum toxin.     

Assessment of biostimulation and bioaugmentation for removing chlorinated volatile organic compounds from groundwater at a former manufacture plant

Site in a former chemical manufacture plant in China was found contaminated with high level of chlorinated volatile organic compounds (CVOCs). The major contaminants chloroform (CF), 1,2-dichloroethane (1,2-DCA) and vinyl chloride (VC) in groundwater were up to 4.49 × 10⁴, 2.76 × 10⁶ and 4.35 × 10⁴ μg/L, respectively. Ethene and methane were at concentrations up to 2219.80 and 165.85 μg/L, respectively. To test the hypothesis that the CVOCs in groundwater at this site could be removed via biodegradation, biomarker analyses and microcosm studies were conducted. Dehalococcoides 16S rRNA gene and VC-reductase gene vcrA at densities up to 1.5 × 10⁴ and 3.2 × 10⁴ copies/L were detected in some of the groundwater samples, providing strong evidence that dechlorinating bacteria were present in the aquifer. Results from the microcosm studies showed that at moderate concentrations (CF about 4000 μg/L and 1,2-DCA about 100 μg/L), CF was recalcitrant under natural condition but was degraded under biostimulation and bioaugmentation, while 1,2-DCA was degraded under all the three conditions. At high concentration (CF about 1,000,000 μg/L and 1,2-DCA about 20,000 μg/L), CF was recalcitrant under all the three treatments and 1,2-DCA was only degraded under bioaugmentation, indicating that high concentrations of contaminants were inhibitory to the bacteria. Electron donors had influence on the degradation of contaminants. Of the four fatty acids (pyruvate, acetate, propionate and lactate) examined, all could stimulate the degradation of 1,2-DCA at both moderate and high concentrations, whereas only pyruvate and acetate could stimulate the degradation of CF at moderate concentration. In the microcosms, the observed first-order degradation rates of CF and 1,2-DCA were up to 0.12 and 0.11/day, respectively. Results from the present study provided scientific basis for remediating CVOCs contaminated groundwater at the site.     

Effects of rumen-protected methionine on plasma amino acid concentrations during a period of weight loss for late gestating beef heifers

This study determined changes in plasma amino acid concentration in late-gestating (beginning 58 ± 1.02 days prior to calving), primiparous, winter-grazing range heifers receiving wheat middling-based supplement without (CON) or with rumen-protected methionine (MET) to provide 15 g dl-MET each day. Plasma was collected on days ?2 and 0 (start of MET supplementation just prior to individually receiving supplement at 0700 hours). Plasma was sampled again on days 40, 42 and 44 prior to supplementation at 0700 and 1100 hours (4 h after receiving daily supplement). Data were analyzed with cow as the experimental unit. Continuous variables were analyzed by the main effects of treatment, date, or time and their interaction when appropriate. Comparable BW (P = 0.32) and BCS (P = 0.83) over the 44-day metabolism trial were found between both CON- and MET-fed heifers. MET-supplemented heifers had greater (P < 0.01) plasma concentrations of methionine indicating that the rumen-protection technology successfully delivered methionine to the small intestine. Supplementation with rumen-protected dl-MET caused a significant supplement × date interaction for glutamine (P = 0.03), glycine (P = 0.02), methionine (P < 0.01), and serine (P = 0.05). In addition, trends for supplement × date interactions were detected for leucine (P = 0.07), threonine (P = 0.09), valine (P = 0.08), total amino acids (TAA; P = 0.08), non essential amino acids (NEAA; P = 0.08), branched chain amino acids (BCAA; P = 0.08), and glucogenic amino acids (GLUCO; P = 0.08). These results suggest that the BCAA (leucine and valine) were utilized more efficiently with MET supplemented heifers compared to CON supplemented heifers. Plasma AA concentrations for glutamic acid (P < 0.01), histidine (P = 0.01), tyrosine (P < 0.01), and EAA (P < 0.01), all decreased throughout the study. These results further confirm methionine is a limiting amino acid in forage fed late-gestating heifers and further suggests the limitation when grazing dormant range forages as shown by improved utilization of other plasma amino acids when supplemental methionine was provided.     

Determination of oxalate in urine and plasma using reversed-phase ion-pair high-performance liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II)-electrogenerated chemiluminescence detection

Oxalate is quantitated in both urine and plasma samples using reversed-phase ion-pair high-performance liquid chromatography (HPLC) with tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺]-electrogenerated chemiluminescent (ECL) detection. Underivatized oxalate was separated on a reversed-phase column (Zorbax ODS) using a mobile phase of 10% methanol in 100 mM phsophate buffer at pH 7.0. The eluted compounds were combined with a stream of 2 mM Ru(bpy)₃²⁺ at a mixing tee before the ECL flow-cell. In the flow-cell, Ru(bpy)₃²⁺ is oxidized to Ru(bpy)₃²⁺ at a platinum electrode, and reacts with oxalate to produce chemiluminescence (CL). Urine samples were filtered and diluted prior to injection. Plasma samples were deproteinized before injection. A 25-μl aliquot of sample was injected for analysis. Possible interferants, including amino acids and indole-based compounds, present in biological samples were investigated. Without the separation, amino acids interfere by increasing the total observed CL intensity; this is expected because they give rise to CL emission on their own in reaction with Ru(bpy)₃³⁺. Indole compounds exhibit a unique interference by decreasing the CL signal when present with oxalate. Indoles inhibit their own CL emission at high concentration. By use of the indicated HPLC separation, oxalate was adequately separated from both types of interferants, which thus had no effect on the oxalate signal. Urine samples were assayed by both HPLC and enzymatic tests, the two techniques giving similar results, differing only by 1%. Detection limits were determined to be below 1 μM (1 nmol/ml) or 25 pmol injected. The working curve for oxalate was linear throughout the entire clinical range in both urine and plasma.