Analysis of enkephalins, beta-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism

Sensitivity in the 10-100 pg range for enkephalins, beta-endorphin, tyrosine (T), 12 tyrosylglycine (T-G) and tyrosylglycylglycine (T-G-G) was attained by using a high-performance liquid chromatographic (HPLC) method with electrochemical detection which is at least 100 times more sensitive than HPLC with UV detection. The chromatographic conditions on a reversed-phase C18 silica column were 50 mM sodium phosphate buffer (pH 2.1) (A) in acetonitrile-methanol (1:1) (B), isocratic mixture, flow-rate 0.6-1 ml/min, UV detection at 205 nm, electrochemical oxidation potential + 1.25 V. The separation of T, T-G and T-G-G was obtained by using 10% B while the separation of the pentapeptide, enkephalins required 40% B. Separation of enkephalins from beta-endorphin was attained at a shorter retention times did not exceed 15 min. This method can be used to determine tissue levels and pharmacodynamics of enkephalins and beta-endorphin. A highly specific measurement of the different enzymes involved in the metabolism of enkephalin has been achieved.     

HILIC-MS/MS method development for targeted quantitation of metabolites: practical considerations from a clinical diagnostic perspective

The development of targeted assays for polar molecules is a persistent challenge in quantitative metabolite measurement. In addressing these challenges, hydrophilic interaction liquid chromatography (HILIC) has proved to be a valuable, though under-used and poorly understood chromatographic technique. This work has addressed a number of components that are intrinsic in development of a high-throughput, specific and sensitive assay for metabolites using HILIC-MS/MS. Generally accepted HILIC doctrine, such as addition of water to all mobile phases and re-equilibration time, was shown to be flawed under gradient HILIC mode. The effect of non-classical mobile phase additives on HILIC-MS/MS specificity, sensitivity and assay throughput was shown for endogenous metabolites. A broad evaluation of columns and mobile phases for the retention of varied molecular classes was performed, elucidating the empirical nature of HILIC method development. Application of the empirical evaluations performed in the paper was demonstrated by detailing a method development cycle for methylmalonic acid to achieve a highly selective and sensitive HILIC-MS/MS quantitation capable of high-throughput analysis for clinical utility.     

Selective and sensitive determination of bis(7)-tacrine, a high erythrocyte binding acetylcholinesterase inhibitor, in rat plasma by high-performance liquid chromatography-tandem mass spectrometry

The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.     

Micellar and aqueous‐organic liquid chromatography using sub‐2 μm packings for fast separation of natural phenolic compounds

The objective of the present work was to investigate the chromatographic behavior of natural phenolic compounds in micellar and aqueous‐organic LC using a short column packed with 1.8 μm particles. Firstly, the effect of ACN and SDS on elution strength and selectivity was examined by isocratic submicellar (0–30% ACN/5% 1‐butanol/1–6 mM SDS) and micellar (0–30% ACN/5% 1‐butanol/40–60 mM SDS) systems. The varied concentrations of two modifiers in the mobile phases revealed different eluting power. Then, the application of organic modifier gradient was discussed in both submicellar and micellar LC using mobile phases of 4 mM SDS/5% 1‐butanol or 50 mM SDS/5% 1‐butanol containing ACN gradient from 0 to 30%, respectively. For micellar system, the separation was found to be better in gradient than isocratic elution. Additionally, the sensitivity of aqueous‐organic LC was examined. The mobile phase was a mixture of ACN and water employing gradient elution at a flow rate of 0.5 mL/min, with analysis time below 9 min. It was found that separation efficiency was significantly better compared with micellar LC. Besides, the aqueous‐organic LC has been applied to separation of various phenolic compounds in Yangwei granule or Radix Astragali samples.     

Cylindrospermopsin determination using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) as the internal standard

Cylindrospermopsin (CYN) was determined by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) as the internal standard. In the selected ion monitoring of LC/ESI-MS, m/z 414 for CYN and 237 for HEPES were monitored using the negative mode; the retention times of CYN and HEPES were 12.41 and 14.21 min, respectively. CYN was determined from peak area ratios of m/z 414/237. By the treatment of an anion exchange cartridge using a buffer at pH 10.5, CYN was isolated and condensed. No interfering peak was observed. Linearity of this method was observed at the range of 0.10-31.12 ng. Total coefficients of variation were 5.1 and 2.9% at 104 and 1038 μg CYN L⁻¹. The quantitative limit at a signal-to-noise (S/N) ratio of 10 was 0.16 ng.CYN concentration in natural waters is low. CYN in waters should be condensed for determination. This method including the treatment for isolation and condensation of CYN is useful for determination of CYN in environmental and/or drinking waters.     

Determination of histidine and related compounds in rumen fluid by liquid chromatography

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45-90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (microM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4-103.0% within a 1-day study and 95.4-99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 microM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 microM, respectively, before the morning feeding.     

Rapid and simultaneous determination of hexapeptides (Ac-EEMQRR-amide and H2N-EEMQRR-amide) in anti-wrinkle cosmetics by hydrophilic interaction liquid chromatography-solid phase extraction preparation and hydrophilic interaction liquid chromatography with tandem mass spectrometry

A rapid method for the simultaneous determination of Ac-EEMQRR-amide and H(2)N-EEMQRR-amide in cosmetic products was developed and evaluated. This analytical procedure involved extracting samples with 0.1:0.1:85:15 (v:v) trifluoroacetic acid (TFA):formic acid:acetonitrile (ACN):water and determination by hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Samples showing serious ion suppression were further cleaned up using HILIC-SPE prior to HILIC-MS/MS analysis. Stable isotopically labeled peptides, corresponding to the above two peptides, were used as internal standards to correct for loss of recovery and matrix effects. Electrospray ionization (ESI) in the positive mode was used. The linear range was 2.0-1000 ng/mL for Ac-EEMQRR-amide and 25.0-2500 ng/mL for H(2)N-EEMQRR-amide. Thirteen commercial products were analyzed for the two peptides using this method. The amounts of Ac-EEMQRR-amide in the samples ranged from none detected to 42.3 μg/g. H(2)N-EEMQRR-amide was not detected in any of the samples. The recoveries for Ac-EEMQRR-amide and H(2)N-EEMQRR-amide ranged from 85% to 110% and 84% to 119%, respectively, at the spiking level of 30 μg/g.     

Determination of trehalose-6-phosphate in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedlings by hydrophilic-interaction liquid chromatography–mass spectrometry

A hydrophilic-interaction chromatography (HILIC) method coupled to electrospray ionization mass spectrometry (ESI-MS) was developed for the determination of trehalose-6-phophate (Tre6P) in Arabidopsis thaliana seedlings. The method was optimized for MS detection and separation of Tre6P from its isomers, such as sucrose-6-phosphate, by testing eluent pH, type of organic solvent and alkalinizer, and gradient conditions. Tre6P could be resolved from matrix components within 28 min by using a water–acetonitrile gradient (0.2 ml/min) at pH 12 with piperidine as alkalinizer. The method was validated for concentrations between 25 and 4,000 nM Tre6P in A. thaliana seedling extracts. Seedlings were extracted with consecutive liquid-liquid and solid-phase extractions, and analyzed with HILIC-MS. Obtained accuracy (80–120 %) and precision (<24 %) demonstrated the suitability of HILIC-MS for determining Tre6P level variations in plants. The limit of detection (LOD) was 3.5 nM Tre6P in extracts corresponding to 4.1 pmol.g⁻¹ fresh plant weight (FW). This is a considerable improvement with respect to anion-exchange chromatography (AEC)-MS (40 nM) and capillary electrophoresis-MS (80 nM). Furthermore, HILIC-MS analysis times were shorter than with AEC-MS (30 and 60 min, respectively). The applicability of the HILIC-MS method was demonstrated by the analysis of extracts from seedlings grown on medium containing 100 mM sorbitol or trehalose, resulting in mean Tre6P concentrations of 0.2 and 1.9 nmol.g⁻¹ FW, respectively. Similar concentrations were found with AEC-MS. HILIC-MS was also evaluated at a high flow rate (2.0 ml/min). This high-speed method resolved the Suc6P and Tre6P peaks within 3 min yielding a detection limit of 1.3 nM Tre6P.     

Dithizone derivatives as sensitive water soluble chromogenic reagents for the ion chromatographic determination of inorganic and organo-mercury in aqueous matrices

Water-soluble sulfonate and the novel carboxylate analogues of dithizone, combined with ion interaction chromatography on a Dionex Acclaim 120 C18 silica column (250 x 4.6 mm id) with an eluent consisting of 10 mM tetrabutylammonium bromide and 60:40 methanol:water, have been developed as highly sensitive chromogenic ligands for the quantitative isocratic determination of inorganic and organo-mercury compounds in aqueous matrices in under 12 min. Using an optimised post column reagent system containing 0.65 mM dye, 0.5% Triton X-100 and 50 mM sodium hydroxide, good linearity (0-7.5 mg L(-1) R2 > 0.999), reproducibility using peak area measurements (RSD 0.69-1.38%, n = 8), and limits of detection (4-12 microg L(-1)) were achieved for methyl mercury, inorganic mercury and phenyl mercury.     

Electrophoretic determination of calystegines A3 and B2 in potato

Potatoes, members of the Solanaceae plant family, contain calystegines, water-soluble nortropane alkaloids, which are biologically active as glycosidase inhibitors. The content of calystegines A(3) and B(2) in different varieties of potato and in various parts of the tubers (whole potato, peel, flesh, and sprouts) were analysed by new capillary zone electrophoresis and capillary isotachophoresis methods and by the routine GC method. The optimized background electrolyte for capillary zone electrophoretic analysis was mixture of 20 mM histidine, 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and 20% (v/v) methanol in demineralized water. Calystegines were detected by indirect UV detection at 210 nm. A clear separation of calystegines from other components of the methanolic sample extract was achieved within 4 min. The electrolytes for isotachophoretic analysis consisted of 5 mM NH(4)OH, 10 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 0.1% hydroxyethylcellulose and 20% (v/v) methanol in demineralized water (leading) and 5 mM histidine+10 mM acetic acid+20% (v/v) methanol in demineralized water (terminating). Calystegines were separated within 20 min and detected by a conductimeter. Method characteristics of both zone electrophoresis and isotachophoresis, i.e., linearity (10-100 ng/microl and 1-10 ng/microl), accuracy (recovery 96+/-5% and 98+/-4%), intra-assay repeatability (4.2% and 3.5%), and detection limit (3 and 0.4 ng/microl) were evaluated. Simple sample preparation, sufficient sensitivity, speed of analysis, and low running cost are important attributes of the electrophoretic methods. The overall results of electrophoretic methods were comparable with GC.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for rapid and sensitive analysis of two new bioactive reagents using dynamic covalent coating

A simple, rapid, selective, and sensitive micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection (LIF) method was developed, using hexamethyldisilazane (HMDS) as dynamic covalent coating (DCC), for the analysis of two new bioactive agents N-n-hexyl-N'-(sodium p-aminobenzenesulfonate) thiourea (HXPT) and N-n-undecyl-N'-(sodium p-aminobenzenesulfonate) thiourea (UPT) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. MEKC methods both not using DCC and using DCC were investigated. In a series of optimization steps, DCC and a running buffer of 20 mM Na2B4O7 + 16 mM SDS + 8% acetonitrile were applied for determination of the derivatives. Linear relationships for HXPT and UPT were obtained in the range of 5 to 100 microM (correlation coefficient: 0.9986 for HXPT, 0.9978 for UPT), and the detection limits for HXPT and UPT were 16.5 and 39.0 ng mL(-1). The sensitivity was improved over that of fluorescence spectroscopy methods. The method was applied to the analysis of the two reagents in lab water waste with recoveries in the range of 95.6-107.5%.     

Determination of 5-nitroimidazoles and metabolites in environmental samples by micellar electrokinetic chromatography

A method based on micellar electrokinetic chromatography (MEKC) with UV detection has been developed for the determination of nine 5-nitroimidazoles (5-NDZs), including metabolites in river water samples. Due to the relative insensitivity of UV detection in MEKC, a solid-phase extraction (SPE) method has been proposed that preconcentrates water samples fiftyfold and cleans them up off-line. An on-line preconcentration approach based on sweeping and the use of an extended light path fused-silica capillary (64.5?cm?×?50?μm i.d., 56?cm effective length) was also found to improve the sensitivity of the method. Separation was carried out in <21?min using 20?mM phosphate buffer (pH 6.5) and 150?mM SDS as the background electrolyte (BGE). The temperature of the capillary was kept constant at 20°C, a voltage of 25?kV was applied (normal mode), and a detected wavelength of 320?nm was utilized. Hydrodynamic injection (50?mbar for 15?s) of the samples, which were dissolved in 20?mM phosphate (pH 6.5), was employed. The limits of detection were lower than 1.1?μg?L(-1). Recoveries of >80% from spiked river water samples were obtained for most of the analytes at three different concentration levels with acceptable precision. This method could provide an efficient and economical alternative to the use of chromatographic methods to monitor nitroimidazole residues, thus supplementing the relatively few methods available for the analysis of these compounds in environmental samples.     

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.     

Simultaneous quantification of methylene blue and its major metabolite,azure B,in plasma by LC‐MS/MS and its application for a pharmacokinetic study

A simple and sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of Olanzapine in rat brain using liquid chromatography with coulometric detection and a rapid solid-phase extraction procedure

A sensitive and selective method was developed for the determination of the antipsychotic drug Olanzapine levels in rat brain tissue, based on HPLC with electrochemical detection. The analyses were carried out on a C8 reversed phase column (150 mm x 4.6 mm, 5 microm), using a mobile phase composed of methanol and a phosphate buffer (44.0 mM, pH 3.5), containing triethylamine (21:79, v/v), flowing at 1.2 mL min(-1). A high sensitivity coulometric detection analytical cell containing two flow-through low volume working electrodes was used: electrode 1 was set at +0.350 V and electrode 2 at -0.200 V. Olanzapine, administered to rats in different doses or in different times, was extracted from tissue homogenate of either the whole brain or specific areas (cortex, hyppocampus, nucleus striatum) with a rapid solid phase extraction procedure (SPE) on Oasis HLB cartridges. The method provided a high extraction yield of Olanzapine and internal standard (2-methylolanzapine) from brain tissue homogenate with absolute recovery values higher than 90.0%. The detector response was linear over a concentration range of 0.2-100.0 ng mL(-1) of Olanzapine. The limit of quantification (LOQ) was 0.2 ng mL(-1). Precision results, expressed by the intra-day and the inter-day relative standard deviation values, were satisfactory, better than 4.6%. Accuracy was satisfactory as well. This method proved to be suitable for the analysis of Olanzapine in rat brain tissues and for the study of distribution and pharmacokinetics of Olanzapine in rat brain after a single treatment with the antipsychotic drug.     

Quantification of doxazosin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry

Hydrophilic interaction LC with MS/MS (HILIC-MS/MS) was described as a rapid, sensitive, and selective method for the quantification of doxazosin in human plasma. Doxazosin and cisapride (internal standard) were extracted from human plasma with ethyl acetate at alkaline pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of ACN/ammonium formate (100 mM, pH 4.5) (93:7 v/v). The analytes were detected using an ESI MS/MS in the selective-reaction-monitoring mode. The standard curve was linear (r = 0.9994) over the concentration range of 0.2-50 ng/mL. The LOQ for doxazosin was 0.2 ng/mL using 100 microL plasma sample. The CV and relative error for intra- and interassay at four QC levels were 3.7-8.7% and 0.0-9.8%, respectively. The matrix effect for doxazosin and cisapride were practically absent. The recoveries of doxazosin and cisapride were 67.4 and 61.7%, respectively. This method was successfully applied to the pharmacokinetic study of doxazosin in humans.     

Isocratic reversed phase high performance liquid chromatography determination of twelve natural corticosteroids in serum with on-line ultraviolet and fluorescence detection

An isocratic reversed phase high performance liquid chromatography procedure utilizing ultraviolet and fluorescence detectors linked in series is described for the analysis of cortisone (E), cortisol (F), corticosterone (B), 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), androstenedione (A), testosterone (T), 17-hydroxyprogesterone (17-OHP), progesterone (P), estriol, estradiol, estrone, prednisone acetate and dexamethasone acetate in serum. Serum specimens were extracted with ethyl ether. The optimized mobile phase was methanol + tetrahydrofuran + water (26:18:56, v/v/v). A Shim-pack ODS column was used. The recoveries were 80 to 103%. Intra- and inter-day coefficient of variance were less than 8%. The detection limit is 0.5 pmol per injection volume for estriol, estradiol, E, F and B; 1 pmol for S, A, DOC and estrone; 2 pmol for T and 17-OHP; and 4 pmol for P. Serum from normal subjects and patients with congenital adrenal hyperplasia due to 21- or 17-hydroxylase deficiency were measured, as well as samples of maternal and umbilical cord serum.     

Biosynthesis of 15N-labeled cylindrospermopsin and its application as internal standard in stable isotope dilution analysis

Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[¹⁵N₅]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na¹⁵NO₃-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103 %; the limit of quantification was 15 ng/L in freshwater and 50 μg/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8 ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0 μg/kg fresh weight and 23.9 μg/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis.      

Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography.

Assessment of lactate metabolism is of particular interest during exercise and in disease states such as diabetes, shock, and absorptive abnormalities of short-chain fatty acids by the colon. We describe an analytical method that introduces radio-active tracers and high-performance liquid chromatography (HPLC) to simultaneously analyze concentrations and specific activities (SAs) of plasma lactate. The HPLC conditions included separation on a reversed-phase column (octadecylsilane) and an isocratic buffer (30% acetonitrile in water). [3H]Acetate served as an internal standard. Lactate and acetate were extracted from plasma samples with diethyl ether following a pH adjustment to less than 1.0 and back-extracted into a hydrophilic phase with sodium carbonate (2 mM, pH greater than 10.0). Lactate is detected in the ultraviolet range (242 and 320 nm) by derivatization with alpha-bromoacetophenone. Control plasma samples were studied after an overnight fast for precision and analytical recovery. Calibration curves were linear in the range 0.18-6.0 mM (r = 0.92). The precision was 3% and the analytical recovery was 87%. The detection limit of the method was 36 pmol. Determination of lactate metabolism was performed in a patient with chronic congestive heart failure who was administered primed-continuous L-[U-14C]lactate (10 microCi bolus and 0.3 microCi/min continuously) during a 60-min rest period. Mean arterial lactate concentration and SA were 1.69 +/- 0.2 mM and 253.8 +/- 22 dpm/mumol, respectively. Systemic lactate turnover was 25.65 mumol/kg per min. Lactic acid systemic turnover, organ uptake and release rates can be accurately determined by isocratic HPLC.     

Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC-MS/MS method for L-arginine and its dimethylated derivatives detection

Asymmetric N(G),-N(G)-dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free L-arginine (Arg), ADMA, and symmetric N(G),-N(G')-dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to 30 degrees C.