Determination of plasma oxalic acid by high-pressure liquid chromatography

A new specific and sensitive method for determination of oxalic acid in plasma by High Performance Liquid Chromatography (HPLC) is described. The plasma sample is deproteinized by ultrafiltration. The oxalic acid in the ultrafiltrate is purified by precipitation with CaCl2, new dilution of calcium oxalate precipitate, oxalic acid extraction with diethyl-ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm with a retention time of 3.20 minutes. The method shows a mean recovery value of 82.11, with an intra-run and between-run CV values of 2.54 and 6.95 respectively. The oxalic acid measured in plasma by this method is 291 +/- 89 micrograms/100 ml plasma ultrafiltrate, in 16 normal subjects.     

Specific and rapid assay of urinary oxalic acid using high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed to determine the levels of oxalic acid in the urine. This acid was extracted from urine with tri-n-butyl phosphate and converted into the fluorescent derivative by esterification with 9-anthryldiazomethane (ADAM). The reaction mixture containing the oxalic acid derivative can be directly chromatographed on HPLC using octadecylsilane reverse-phase column monitoring with a fluorophotometric detector. A linear relationship was observed in the range from 1 to 100 micrograms/ml of standard oxalic acid dissolved in saline. Disease-free Japanese adults excrete 23.8 +/- 9.0 mg (mean +/- SD) of oxalic acid per day. This method should prove valuable for routine measurements of urinary oxalic acid as it is accurate, simple, and specific.     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels

A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o-phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C₈ column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.     

Large-scale HPLC purification of calbindin D9k from porcine intestine

Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.     

Determination of fenofibric acid in human plasma using automated solid-phase extraction coupled to liquid chromatography

The pharmacokinetic studies of fenofibrate require a rapid, selective and robust method to allow the determination of fenofibric acid, its active metabolite, in different biological matrixes (such as plasma, serum or urine). A new fully automated method for the determination of fenofibric acid in plasma has been developed, which involves the solid-phase extraction (SPE) of the analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase HPLC with UV detection. The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with octadecyl silica was first conditioned with methanol and pH 7.4 phosphate buffer. A 0.8-ml volume of diluted plasma sample containing the internal standard (sulindac) was then applied on the DEC. The washing step was performed with the same buffer (pH 7.4). Finally, the analytes were successively eluted with methanol (1.0 ml) and 0.04 M phosphoric acid (1.0 ml). After a mixing step, 100 μl of the resultant extract was directly introduced into the HPLC system. The liquid chromatographic (LC) separation of the analytes was achieved on a Nucleosil RP-8 stationary phase (5 μm). The mobile phase consisted of a mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was monitored photometrically at 288 nm. The method developed was validated. In these conditions, the absolute recovery of fenofibric acid was close to 100% and a linear calibration curve was obtained in the concentration range from 0.25 to 20 μg/ml. The mean RSD values for repeatability and intermediate precision were 1.7 and 3.9% for fenofibric acid. The method developed was successfully used to investigate the bioequivalence between a micronized fenofibrate capsule formulation and a fenofibrate Lidose™ formulation.     

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 degrees C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 microl plasma. Detection limits were 0.1 microg/ml for MPA and AcMPAG, and 2.0 microg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.     

Determination of deoxynivalenol (DON) in blood, bile, urine and excrement samples from swine using immunoaffinity chromatography and LC-UV-detection

A work up procedure is described by which DON concentrations in blood, bile, urine and excrements from swine can be quantified by HPLC and UV- detection at λ = 220 nm. The central step thereby is the purification and concentration of DON by means of an immunoaffinity column. While, in our experiments, the quantification of DON in blood and urine was straightforward an additional purification step by a preparative HPLC run prior to immunoaffinity chromatography was needed when bile and excrements were investigated. However, when low DON concentrations in blood and urine are expected, a preparative HPLC run prior to immunoaffinity chromatography is recommended as well, because larger amounts of sample materials should be analyzed and more impurities interfere with the column proteins. In our study, using spiked samples, recoveries ranged from 75—90% and limits of detection were 0.01 to 0.02 μg/ml.     

Determination of IAA and ABA in the same plant sample by a widely applicable method using GC-MS with selected ion monitoring

A method for the purification and subsequent quantification of indole-3-acetic acid (IAA) and abscisic acid (ABA) from the same sample of highly pigmented green tissue has been developed and tested in several species. Solvent partitioning and high-performance liquid chromatography (HPLC) were used for purification. Separate fractions from HPLC-containing IAA and ABA were analyzed by gas chromatography-mass spectrometry (GC-MS) using selected-ion monitoring (SIM). Isotope dilution was used to correct for incomplete recovery. Results are presented for tissue samples from 11 different species and five different plant organs. The method can be completed, for both IAA and ABA, for two samples in 8 h by an experienced technician. IAA and ABA were the dominant peaks in the gas chromatograms from HPLC-purified samples, and amounts of about 1 ng can be detected. The extract was partitioned into an aqueous solution of pH 9.5, a step suspected of ester hydrolysis. By analyzing samples known to contain esters of IAA and ABA and comparing the results with methods which excluded this step, we have shown that this partitioning does not result in erroneously high values due to ester hydrolysis. A direct comparison of the method with one in which HPLC was not employed indicates that our method measures IAA and ABA in samples in which these compounds are not detectable when HPLC is omitted. Thus, HPLC is an essential purification step for samples where contaminating compounds co-purify with IAA and ABA through the solvent-partitioning steps.     

Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on oxalate oxidase and SIRE technology

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.     

Simultaneous determination of 1- and 2-naphthol in human urine using on-line clean-up column-switching liquid chromatography-fluorescence detection

We developed a new 3-D HPLC method for on-line clean-up and simultaneous quantification of two important naphthalene metabolites, 1-naphthol and 2-naphthol, in human urine. Except an enzymatic hydrolysis no further sample pre-treatment is necessary. The metabolites are stripped from urinary matrix by on-line extraction on a restricted access material pre-column (RAM RP-8), transferred in backflush mode onto a silica-based CN-(cyano)phase column for further purification from interfering substances. By another successive column switching step both analytes are transferred with a minimum of overlapping interferences onto a C12 bonded reversed phase column with trimethylsilyl endcapping where the final separation is carried out. The entire arrangement is software controlled. Eluting analytes are quantified by fluorescence detection (227/430 nm) after an external calibration. Within a total run time of 40 min we can selectively quantify both naphthols with detection limits in the lower ppb range (1.5 and 0.5 microg/l for 1- and 2-naphthol, respectively) with excellent reliability (ensured by precision, accuracy, matrix-independency and FIOH quality assurance program participation). First results on a collective of 53 occupationally non exposed subjects showed mean levels of 11.0 microg/l (1-naphthol) and 12.9 microg/l (2-naphthol). Among smokers (n=21) a significantly elevated mean level of urinary naphthols was determined (1-naphthol: 19.2 microg/l and 2-naphthol: 23.7 microg/l) in comparison to non smokers (n=32; 1-naphthol: 5.6 microg/l, 2-naphthol: 5.6 microg/l).     

Two-dimensional high-performance liquid chromatographic determination of 5-hydroxyindoleacetic acid and homovanillic acid in urine

The biomedically and neurochemically important compounds 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) have been simultaneously determined in human urine after reverse-phase two-dimensional high-performance liquid chromatography. A 10-fold-diluted urine sample (20 microliters) is first separated on a C18 column (30 X 0.39 cm) using an 85% pH 6.0 phosphate buffer/15% methanol solvent system. The elution volume containing both 5-HIAA and HVA (Rt approximately 3 min) is collected. Recoveries (mean +/- SD) for this purification step, which is monitored using fluorometric detection, were usually above 90%. After acidification of the approximately 2 ml collected fraction, 100 microliters is reinjected on a C18 column and separated (Rt: 5-HIAA, 4 min; HVA 5.5 min) using an 80% pH 3.5 phosphate buffer/20% methanol mobile phase. The compounds are determined by flow-through amperometry with absolute detection limits of approximately 25 pg. Both 5-HIAA and HVA are well resolved from other electroactive species present and are easily determined at normal and greatly reduced concentrations in human urine.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

Determination of laquinimod in plasma by coupled-column liquid chromatography with ultraviolet absorbance detection

Laquinimod is an immunomodulator that is currently in clinical trials. For pharmacokinetic and toxicokinetic studies in animals and humans a sensitive and accurate bioanalytical method was required. In this paper a bioanalytical method for the determination of laquinimod by liquid chromatography is described. After a protein precipitation step the plasma sample was injected onto a coupled-column HPLC system. After further purification from macromolecules on a short restricted access material C(18) column the analyte was transferred to a reversed-phase C(18) analytical column and separated from interfering substances. The analyte was detected by UV detection. The method was validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 0.75 micromol/L, the intermediate precision was 1.8-3.6% (C.V.) and the accuracy was 97.7-114.7%. In conclusion, the method was found to perform well and is suitable for use in pharmacokinetic and toxicokinetic studies.     

Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples

A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile-methanol-0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 microl) of the supernatant was injected into the HPLC system linked to a Waters XTerra RP(18) column (150 mm x 4.6 mm i.d., particle size 5 microm). The compound was eluted by a mixture of acetonitrile-methanol-0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50-1200 ng/ml and 1200-12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from -2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration-time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces.     

药用级微生物谷氨酰胺转胺酶的纯化工艺研究

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高 选择性和高载量地捕获,再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。     

High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C₁₈ solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5 · 10⁻⁴ M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.     

Simultaneous determination of oxalic and citric acids in urine by high-performance liquid chromatography

A simple method for the simultaneous determination of oxalic and citric acids has been developed using reversed-phase HPLC. An aqueous solution containing potassium dihydrogenphosphate (0.25%) and tetrabutylammonium hydrogensulphate (2.5 mmol) at pH 2.0 was used as mobile phase. Under these conditions both the components were well resolved and detected at 210 nm. The recovery for oxalic and citric acids was 97% and 102%, respectively. The method presented here was applied to urine specimens of a large number of urolithic patients and control subjects. Because of the simplicity of the method its application provides better means of monitoring the concentration of oxalic and citric acids in the formation of renal calculi.     

Purification of N-terminal hexapeptide of big gastrin from human urine

We previously demonstrated that extremely high amounts of N-terminal big gastrin (G-34) fragments are excreted in human urine and three of them are N-terminal octa-, nona-, and decapeptide of G-34. Our subsequent examination revealed that there exists a considerable amount of another N-terminal G-34 fragment in urine, less hydrophobic than the three peptides. We purified this fragment from urine of an achlorhydric patient and determined the structure: less than Glu-Leu-Gly-Pro-Gln-Gly. The purification was carried out by Sep-Pak C18 cartridges, Sephadex G-25, and reverse phase HPLC. The structure was determined by a combination of amino acid analysis, amino acid sequence analysis, and mass spectral analysis. N-terminal hexapeptide of G-34 is the second richest component of urinary N-terminal G-34 fragments next to N-terminal octapeptide of G-34 in normal subjects.     

Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate

The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid–liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5–8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.