血浆中极长链脂肪酸的毛细管气相色谱分析

建立了血浆中极长链脂肪酸毛细管气相色谱法，并用于测定了１０例正常人和４例临床拟诊为肾上腺白质营养不良（ＡＬＤ）病人血浆中极长链脂肪酸，其中一例肯定了ＡＬＤ的临床诊断。     

Isolation and preparative purification of microcystin variants

Preparative reversed-phase liquid chromatography was successfully used to purify two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. The separation protocol involved extraction of lyophilized cells by methanol, isolation and concentration by solid-phase extraction, and purification by reversed-phase HPLC. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C18 stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column. The microcystins were quantified by HPLC and enzyme-linked immunosorbent assay. A method to remove microcystins rapidly and economically from the cyanobacterial process waste is also described.     

Simultaneous analysis of diazepam and its metabolites in rat plasma and brain tissue by HPLC-UV and SPE

Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to “normal” rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 μL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.     

Determination of 4-nonylphenol and 4-octylphenol in human blood samples by high-performance liquid chromatography with multi-electrode electrochemical coulometric-array detection

Alkylphenols can affect human health because they disrupt the endocrine system. In this study, an analytical method for determining trace amounts of 4-nonylphenol (NP) and 4-octylphenol (OP) in human blood samples was developed. Reversed-phase HPLC with multi-electrode electrochemical coulometric-array detection was used for the determination of NP and OP in plasma and serum samples prepared with a solid-phase extraction method. The separation was achieved using an isocratic mobile phase of 0.7% phosphoric acid-acetonitrile with a C18 reversed phase column. The detection limits of NP and OP were 1.0 and 0.5 ng ml-1, respectively. The recoveries of NP and OP added to human plasma samples were above 70.0% with a relative standard deviation of less than 15.5%. The method was found to be applicable to the determination of NP and OP in various human blood samples such as serum and plasma.     

Multiwalled carbon nanotubes as sorbent for on-line coupling of solid-phase extraction to high-performance liquid chromatography for simultaneous determination of 10 sulfonamides in eggs and pork

The sulfonamides (SAs) have been widely used as effective chemotherapeutics and growth promoters in animals' feeding, but their residues could be a potential danger to human health due to their carcinogenic potency and possible antibiotic resistance. Development of a simple and sensitive method for the determination of SAs residues in food of animal origin, therefore, is of great significance. An on-line solid-phase extraction (SPE) method using multiwalled carbon nanotubes as sorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous determination of 10 sulfonamides (SAs) in eggs and pork was developed. The adsorptive potential of carbon nanotubes for solid-phase extraction of sulfonamides was investigated for the first time in the present paper. To on-line interface solid-phase extraction with HPLC, a conventional sample loop on the six-port injector valve of the HPLC was replaced by a preconcentration column packed with carbon nanotubes. The analytes in water solution were preconcentrated onto the preconcentration column and subsequently eluted with mobile phase of methanol-water (22:78). The developed on-line solid-phase extraction method for HPLC permitted the current HPLC separation and the next preconcentration proceeded in parallel, and thus allows one determination finished within 35 min. The RSD of 10 SAs for nine replicate measurements of a standard mixture of 1 microgl(-1) were in the range of 2.5-7.8%. The method was applied to the determination of trace sulfadiazin (SDZ), sulfamerazine (SMR), sulfadimidine (SDMD), sulfathiazole (STZ), sulfamoxol (SMO), sulfamethizole (SMT), sulfamethoxypyridazine (SMP), sulfachlorpyridazine (SCP), sulfadoxin (SDX) and sulfisoxazole (SIA) in eggs and pork. The results indicated that the proposed method was simple, cost-effective and sensitive.     

Exogenous Addition of 25-Hydroxycholesterol Reduces Level of Very Long-Chain Fatty Acids in X-Linked Adrenoleukodystrophy

X-Linked adrenoleukodystrophy (X-ALD) is a severe metabolic disorder characterized by the accumulation of very long-chain fatty acids (VLCFAs). Recently, we demonstrated that levels of 25-hydroxycholesterol (25-HC) and cholesterol 25-hydroxylase (CH25H) were found to be elevated in X-ALD. Herein, we report that the exogenous addition of 25-HC significantly reduces C26:0 levels in X-ALD patient-derived fibroblasts and oligodendrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from X-ALD patients. Moreover, 25-HC treatment was found to down-regulate the expression of ELOVL1, a key enzyme for the synthesis of C26. In addition, activation of liver X receptor (LXR), a molecular target of endogenous 25-HC, also reduced C26:0 level. The reduction of C26:0 levels by 25-HC treatment might result, at least partially, from the decrease of ELOVL1 expression as well as the activation of LXR. Our findings could provide a better understanding of the role of 25-HC in X-ALD and useful information to find therapeutic agents to treat X-ALD.     

Improved high-performance liquid chromatographic method for the determination of coenzyme Q10 in plasma.

Coenzyme (Co) Q10 was dissociated from lipoproteins in plasma by treatment with methanol and extraction with n-hexane. Subsequent clean-up on silica gel and C18 solid-phase extraction cartridges with complete recovery (99 +/- 1.2%) produced a clean extract. High-performance liquid chromatographic (HPLC) separation was performed on a C18 reversed-phase column. Three simple, rapid procedures are presented: HPLC with final UV (275 nm) detection, a microanalysis utilizing a three-electrode electrochemical detector and a microanalysis with column-switching HPLC and electrochemical detection. The methods correlate very well with classical ethanol-n-hexane extraction with UV detection. The identity and purity of the Co Q10 peak were investigated and the resulting methods were concluded to be suitable for total plasma Co Q10 determination. The average level in healthy subjects was 0.80 +/- 0.20 mg/l; the minimum detectable Co Q10 plasma level was 0.05 and 0.005 mg/l for UV and electrochemical detection, respectively. The methods were applied to many samples and the plasma Co Q10 reference values for healthy subjects, athletes, hyperthyroid, hypothyroid and hypercholesterolaemic patients are given.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Simultaneous analysis of hydrochlorothiazide, triamterene and reserpine in rat plasma by high performance liquid chromatography and tandem solid-phase extraction

A tandem solid-phase extraction method (SPE) of connecting two different cartridges (C(18) and MCX) in series was developed as the extraction procedure in this article, which provided better extraction yields (>86%) for all analytes and more appropriate sample purification from endogenous interference materials compared with a single cartridge. Analyte separation was achieved on a C(18) reversed-phase column at the wavelength of 265 nm by high-performance liquid chromatography (HPLC). The method was validated in terms of extraction yield, precision and accuracy. These assays gave mean accuracy values higher than 89% with RSD values that were always less than 3.8%. The method has been successfully applied to plasma samples from rats after oral administration of target compounds.     

Selective determination, in plasma, of artemether and its major metabolite, dihydroartemisinin, by high-performance liquid chromatography with ultraviolet detection.

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the determination of artemether and its major metabolite dihydroartemisinin in plasma has been developed. It involves extraction of plasma with dichloromethane, solid-phase separation of the two analytes and acid decomposition prior to chromatography on a C18 Spherisorb column with a mobile phase of acetonitrile-water (50:50, v/v). Run time is 30 min. The assay satisfies all of the criteria required for use in clinical pharmacokinetic studies.     

High-performance liquid chromatographic determination of naltrexone in plasma of hemodialysis patients

A simple, sensitive, selective and reliable reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection is described for the determination of naltrexone in plasma samples. Naltrexone and the internal standard, naloxone, were isolated from plasma either with a liquid-liquid extraction method using ethyl acetate or with a solid-phase extraction method using Sep-Pack C18 cartridge before chromatography. The extracts were dried under a stream of nitrogen and the samples were reconstituted in the mobile phase, then 20 microL were injected on a Waters Symmetry C18 column (5 microm particle size, 4.6 x 150 mm). The mobile phase consisted of 0.06% triethylamine (pH 2.8)-acetonitrile (92:8, v/v) pumped at 1 mL/min. The peak-area ratio versus plasma concentration was linear over the range of 10-500 ng/mL and the detection limit was less than 8 ng/mL. Quantification was by ultra-violet detection at 204 nm. The present method was applied to the determination of the plasma concentration of naltrexone in dialyzed patients. Patients (n = 8) with severe generalized pruritus received 50 mg of naltrexone orally per day for 2 weeks. The variability in the therapeutic response in treated patients required plasma concentration investigations of this opioid antagonist.     

Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana)

HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.     

Analysis of plasma free fatty acid cyanomethyl derivatives by GC-NPD for the diagnosis of mitochondrial fatty acid oxidation disorders

Summary Early diagnosis of fatty acid oxidation (FAO) disorders is important to reduce severe morbidity and mortality. Although analysis of plasma free fatty acids (FFAs) is frequently performed using stable isotope-dilution gas chromatography-mass-spectrometry (GC-MS), there are institutions where the required instrumentation is not available to support a rapid work-up of acutely ill patients. For this reason, we have developed a novel cyanomethyl derivatization method for FFAs which is followed by GC analysis of the resulting esters using nitrogen-phosphorus detector (NPD) for the rapid diagnosis of mitochondrial fatty acid oxidation disorders. FFAs were extracted from plasma and derivatized to the cyanomethyl ester by heating with bromoacetonitrile at 60°C for 30 min GC-NPD analysis was then performed. The mean recoveries of C6:0-C18:0FFAs were between 87% abd 96%. The method detection limits (S/N=3) were 0.1–0.5 ng for C6:0-C14:0 FFAs, and 0.001–0.01 ng for C16:0-C18:0 FFAs. We succesfully performed differential diagnosis of representative FAO disorders from the confimed patient's plasmas. This simple method offers cost-effective and time-saving alternative to GC-MS for the biochemical diagnosis of selected FAO disorders.     

Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method.

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C2 ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10-2500 ng/ml in plasma with coefficients of variation < or = 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.     

High-performance liquid chromatographic determination of mitoxantrone in liposome preparations using solid-phase extraction and its application in stability studies

A method for the determination of the entrapped mitoxantrone in liposome preparations was developed. The method consists of a solid-phase extraction procedure followed by HPLC analysis. A C₁₈ cartridge was used for solid-phase extraction and 0.5M methanolic HCl was used for elution. The extraction demonstrated a good separation of the mitoxantrone from the phospholipid. A C₁₈ column and a mobile phase containing acetonitrile-0.01 M monopotassium phosphate (40:60) with the pH adjusted to 3.0 with orthophosphoric acid were employed. The detection wavelength was 242 nm. The HPLC method was stability indicating and was applied to determine the degradation of the entrapped mitoxantrone in liposomes. A pseudo-first-order reaction was found for the degradation of the entrapped mitoxantrone. The half-life of the mitoxantrone decreased with increasing pH of the medium. The results demonstrate that the proposed method is satisfactory for the determination of the stability of mitoxantrone in liposome preparations.     

Determination of polar impurities in biodiesels using solid-phase extraction and gas chromatography-mass spectrometry

This paper reports on a method for development and validation for simultaneous characterization and determination of oxygenated polar impurities--free fatty carboxylic acids (FFAs), partial glycerides (monoacylglycerides, MGs), residual glycerol and free sterols--in various biodiesels based on the combination of solid-phase extraction (SPE), silylation and GC/MS technologies. The effects of various SPE and silylation conditions on the method recoveries were evaluated. Using this integrated SPE-GC/MS method, 38 target polar compounds (13 FFAs, 17 glycerides and 8 sterols) in 9 biodiesels derived from 4 different feedstocks were successfully separated and quantified. It was found that the carbon chain length of FFAs was ranged from C(6) to C(24), with C(16) and C(18) being the most abundant in all biodiesels. The total FFAs concentration was consistent with the acid values (AVs) measured by standard method ASTM D974-04. MG congeners with carbon number of 18 (mono-C18) were most abundant in the biodiesel samples, followed by mono-C(16) and free glycerol. β-Sitosterol and campesterol were found to be the prevailing phytosterols in all pure vegetable oil-based biodiesels, while brassicasterol and stigmasterol was only significant in the biodiesel from canola oil and soybean oil, respectively, and abundant cholesterol was only detected in animal fat-based biodiesels.     

正相高效液相色谱-手性配位基交换流动相法测定人血清中的甲状腺素对映体

 运用正相高效液相色谱 手性配位基交换流动相法 ,通过流动相中添加L 脯氨酸、醋酸铜及三乙胺 ,对人血清中甲状腺素对映体 (D ,L T4)进行了测定。在所建立的色谱条件下 ,D ,L T4异构体得到基线分离。该法用于测定甲状腺机能亢进 (甲亢 )、甲状腺机能减退 (甲低 )患者血清中的D ,L T4的浓度 ,可为临床用药提供理论依据 ,对疾病的治疗起到了积极的作用。     

Simultaneous determination of prednisolone acetate, prednisolone, prednisone, cortisone and hydrocortisone in swine plasma using solid-phase and liquid-liquid extraction techniques

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of prednisolone acetate (PA), prednisolone (PO), prednisone (PN), cortisone and hydrocortisone in swine plasma is described. Extraction of the steroid mixture from swine plasma with dexamethasone as internal standard was accomplished by solid-phase extraction (SPE) or the more traditional liquid-liquid extraction (LLE) techniques. These compounds were analyzed by normal-phase HPLC with ultraviolet detection. Although a detectable sensitivity of 5 ng/ml is achieved by the SPE technique, the practical sensitivity is established as 10 ng/ml. Conversely, the practical sensitivity is 5 ng/ml for all compounds by the LLE technique. Calibration curves were found to be linear between 10 and 500 ng/ml by the SPE technique and between 5 and 100 ng/ml by the LLE technique. The average recovery of the steroids PA, PO and PN at 20 ng/ml is between 70 and 90%. PA is stable for up to 3 h in swine plasma at room temperature (22 degrees C) but is completely converted to PO within 24 h. PA is stable in swine plasma in an ice bath for over 24 h. The usefulness of this analytical technique is demonstrated by the intraperitoneal administration of 125 mg of PA to swine and the quantitative determination of PA, PO and PN in the plasma as a function of time.     

Quantitative and pharmacokinetic analysis of naloxone in plasma using high-performance liquid chromatography with electrochemical detection and solid-phase extraction

In this study we present a method for measuring naloxone in plasma after intravenous and oral administration of naloxone to humans, in order to study its pharmacokinetic profile. The method consists of a solid-phase extraction step followed by detection on a high-performance liquid chromatographic (HPLC) system equipped with an electrochemical dual-electrode detector. The extraction step employs cyanopropyl columns optimized for naloxone extraction to allow for elution of naloxone by the HPLC mobile phase; this eluate is then directly injected in the HPLC instrument. The HPLC system employs a radial compression phenyl column with a mobile phase containing 18% (v/v) acetonitrile and pentanesulfonic acid as ion-pairing agent; this system shows extraordinary high plate counts for naloxone. The detection limit is 3 ng (signal-to-noise ratio = 3) free naloxone per ml plasma. Following intravenous injection of 30 mg naloxone hydrochloride in two subjects, it was possible to determine the free naloxone concentration in the plasma for 8 h, more than four times the half-life of naloxone in plasma in humans.     

Determination of cymoxanil in drinking water and soil using high-performance liquid chromatography

We describe a method for determination of cymoxanil, 1-2-cyano-2-methoxy(iminoacetyl)-3-ethylurea, in drinking water and in soil, using reversed-phase HPLC with UV detection at 240 nm and a mobile phase of acetonitrile-water (30:70, v/v). Fortified water samples (1.0 L) were extracted with solid-phase extraction on Strata X. Soil samples (20 g) were extracted with acetone and the extracts were transferred onto Strata C18E. The recoveries of cymoxanil from water and soil samples were over 85% for each fortification level. The RDS were within the range 1.7-4.1% for water and 0.9-1.2% for soil samples. After optimization of the extraction and separation conditions, the method was validated.