Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

Arsenic speciation based on ion exchange high-performance liquid chromatography hyphenated with hydride generation atomic fluorescence and on-line UV photo oxidation

An on-line method capable of the separation of arsenic species was developed for the speciation of arsenite As(III), arsenate As(V), monomethylarsenic (MMA) and dimethylarsenic acid (DMA) in biological samples. The method is based on the combination of high-performance liquid chromatograph (HPLC) for separation, UV photo oxidation for sample digestion and hydride generation atomic fluorescence spectrometry (HGAFS) for sensitive detection. The best separation results were obtained with an anion-exchange AS11 column protected by an AG11 guard column, and gradient elution with NaH₂PO₄ and water as mobile phase. The on-line UV photo oxidation with 1.5% K₂S₂O₈ in 0.2 mol L^–1 NaOH in an 8 m PTFE coil for 40 s ensures the digestion of organoarsenic compounds. Detection limits for the four species were in the range of 0.11–0.15 ng (20 μL injected). Procedures were validated by analysis of the certified reference materials GBW09103 freeze-dried human urine and the results were in good agreement with the certified values of total arsenic concentration. The method has been successfully applied to speciation studies of blood arsenic species with no need of sample pretreatment. Speciation of arsenic in blood samples collected from two patients after the ingestion of realgar-containing drug reveals slight increase of arsenite and DMA, resulting from the digestion of realgar.     

适用于质谱分析的在线固相萃取除盐方法

建立了一个简单、快速、有效的适用于质谱或液相色谱-质谱联用的在线固相萃取(SPE)高通量除盐方法。方法分为单柱和双柱模式,借助于包含双梯度泵(上样泵/分析泵)、自动进样器和配有十通切换阀的柱温箱的高效液相色谱系统,完成样品的自动化在线除盐。单柱模式通过上样泵实现在SPE柱上进样和除盐,被分析物则保留在SPE柱上;除盐完成后,通过阀切换利用分析泵洗脱富集在SPE柱上的被分析物。双柱模式则在单柱模式基础上增加了1根SPE柱,在色谱管理软件控制下2根SPE柱轮流工作,高效率完成样品的在线除盐。该方法在结合质谱分析蛋白质、多肽等领域具有较好的应用前景。     

Concentration of volatile organic compounds from solvents by sustained chromatographic evaporation

A system for quantitative concentration of volatile organic trace compounds present in organic solvents is described. Evaporation of the solvent is carried out inside a glass capillary tube by the action of a carrier gas, and large volumes can be reduced by a repeated sample injection and a cyclic flow reversal. Best recovery is obtained when a barrier of pure solvent is maintained ahead of the sample during concentration. Four rotary valves are employed for sample and solvent injection and direction of the gas flow. In principle, indefinite sample volumes can be handled, the limit being set by system contaminants. The process was evaluated both off-line and on-line to a gas chromatograph. Concentration of compounds like methylcyclopentane, hexane, and cyclohexane present in pentane in the low nanogram range and subsequent on-line transfer to a gas chromatograph could be performed with a quantitative recovery. The technique was applied to analysis of trace volatiles in drinking water. Detection limits were estimated to be approximately 0.02 ng/L for normal hydrocarbons (FID detection) when concentration of a pentane extract from a one litre water sample was carried out.     

Determination of Cefpodoxime Levels and Cefpodoxime Stability In Human Urine By Direct Injection HPLC with Column-Switching

Abstract

A semi-automated method providing on-line sample extraction and quantitative analysis for cefpodoxime in human urine, injected directly into the HPLC, is reported.

Samples were filtered by the analyst, injected into the HPLC system with an autosampler and loaded onto a 3 cm RP-18 precolumn with a mobile phase consisting of 10% methanol in 0.2% phosphoric acid and then automatically eluted onto a RP-18 analytical column using a mobile phase containing 7% acetonitrile in pH 5.2 sodium acetate buffer. the mean between-day precision of the standards was ± 4.29%. Spiked urine control recovery averaged 96 ± 6% for controls ranging from 1.0 to 20.0 μg/mL. the limit of quantitation for the method was 0.11 μg/mL.     

Trace Enrichment and HPLC Analysis of Chlorophenols in Environmental Samples,Using Precolumn Sample Preconcentration and Electrochemical Detection

Abstract

A HPLC method has been developed for trace analysis of chlorophenols in the 0.2–2 ppb range from spiked water samples. Simple liquid-liquid extraction followed by on-line preconcentration of total mono- and dichlorophenols has been performed using a divinylbenzene-styrene copolymeric sorbent (PRP₁) as packing material for the precolumn. The chlorophenols have been eluted from the precolumn on an analytical column (5μm LiChrosorb RP-18, 12.5 cm × 4 mm) by use of a switching valve system followed by separation. Detection was carried out with an electrochemical detector. The linearity of the detector response has been proved over two orders of magnitude. The detection limit of chlorophenols by means of the electrochemical method is in the lower picogram range. The recoveries of the isomeric chlorophenols from spiked river water samples having initial concentrations of 2ppb are usually 70–90%. The procedure has been applied to drinking water and river water.     

Liquid chromatographic trace enrichment with on-line capillary gas chromatography for the determination of organic pollutants in aqueous samples

Trace enrichment for the GC analysis of a series of chlorinated pesticides and polychlorinated biphenyls (PCBs) in aqueous samples has been achieved through a simple on-line technique involving sorption on an LC micro-precolumn followed by direct elution into a gas chromatograph with hexane. A 5-m retention gap coupled to the capillary GC column served as the recipient of a relatively large sample volume (ca. 100 μl) introduced into the GC. Partially concurrent solvent evaporation during sample introduction allowed a large sample capacity. Recoveries of more than 95% were observed for the majority of the compounds studied. Using 1.0 ml aqueous samples, detection limits of less than 1 ppt were found. The applicability of the developed method was demonstrated for a river water sample.     

Rapid preconcentration method for the determination of azadirachtin-A and -B, nimbin and salannin in neem oil samples by using graphitised carbon solid phase extraction

A simple and rapid method involving solid phase extraction and liquid chromatography for the determination of azadirachtin-A and -B, nimbin and salannin at nanogram levels in neem oil samples is presented. The neem oil samples are defatted and the compounds of interest extracted by mixing the sample with hexane and passing the hexane solution through a graphitised carbon black column. After washing the column with 2 ml of hexane, azadirachtin-A and -B, nimbin and salannin are eluted with 5 ml of acetonitrile and quantified using HPLC with UV detection. The recoveries of azadirachtin-A and -B, nimbin and salannin in fortified oil samples were 97.4-104.7%. The upper limit of quantification is up to 100 micrograms ml-1 without any additional clean-up and with little interference from lipids during the analysis by HPLC. The method was successfully applied to various neem oil samples collected from different locations in India.     

Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection

Various off-line methods for clean-up and sample enrichment are available for the analysis of polycyclic aromatic hydrocarbons (PAHs) in edible oils and fats. These methods consist of laborious and time consuming procedures. This study reports an on-line method using LC-LC coupling. After clean-up of the sample on a donor-acceptor complex chromatography (DACC) column the PAHs are transferred to and separated on an analytical HPLC column. Quantification is carried out with fluorescence detection. The DACC column clean-up is fast and is carried out during the HPLC run of the previous sample. Compared to the traditional methods this automated on-line method saves considerable time and significantly reduces the amount of solvent waste. The method uses common HPLC equipment and its performance has been evaluated.     

Use of open-tubular trapping columns for on-line extraction–capillary gas chromatography of aqueous samples

The applicability of open-tubular trapping columns for on-line extraction–capillary GC analysis is evaluated. The extraction step involves sorption of the analytes from water into the stationary phase of an open-tubular column, removal of the water by purging the trap with nitrogen, and desorption of the analytes with an organic solvent. The effect of swelling of the stationary phase with organic solvents on the retention power of the trap is studied. When using pentane or hexane as swelling agent breakthrough volumes of at least 10 ml can easily be obtained for non-polar compounds. For a number of medium polarity compounds breakthrough volumes of 5 ml can be achieved when chloroform is used as the swelling agent. The required drying time is less than 1 minute. Quantitative desorption requires only 75 μl of organic solvent. Solvent elimination prior to transfer to the GC column is carried out using a PTV injector and a multidimensional GC system. The system is applied for the analyses of river water, urine, and serum samples.     

卷烟主流烟气粒相物中中性化学成分的全二维气相色谱飞行时间质谱(GC×GC-TOF MS)谱图特征识别

建立了全二维气相色谱-飞行时间质谱(GC×GC-TOF MS)分析卷烟主流烟气中中性化学成分的方法。以较长的弱极性柱HP-5MS(50 m×0.2 mm i.d.×0.33μm)作为第一维柱,较短的薄液膜中等极性柱DB-17MS(1.7 m×0.1 mm i.d.×0.1μm)作为第二维柱,对优质烟叶单料卷烟烟气的中性成分进行定性分析,经过人工纠错等分析初步鉴定出匹配度大于700的1 464种成分,重点讨论了中性香味羰基化合物全二维点阵的谱图特征,为烟气和复杂体系的深入研究提供了方法学基础。     

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.     

Arsenic speciation based on ion exchange high-performance liquid chromatography hyphenated with hydride generation atomic fluorescence and on-line UV photo oxidation

An on-line method capable of the separation of arsenic species was developed for the speciation of arsenite As(III), arsenate As(V), monomethylarsenic (MMA) and dimethylarsenic acid (DMA) in biological samples. The method is based on the combination of high-performance liquid chromatograph (HPLC) for separation, UV photo oxidation for sample digestion and hydride generation atomic fluorescence spectrometry (HGAFS) for sensitive detection. The best separation results were obtained with an anion-exchange AS11 column protected by an AG11 guard column, and gradient elution with NaH2PO4 and water as mobile phase. The on-line UV photo oxidation with 1.5% K2S2O8 in 0.2 mol L(-1) NaOH in an 8 m PTFE coil for 40 s ensures the digestion of organoarsenic compounds. Detection limits for the four species were in the range of 0.11-0.15 ng (20 microL injected). Procedures were validated by analysis of the certified reference materials GBW09103 freeze-dried human urine and the results were in good agreement with the certified values of total arsenic concentration. The method has been successfully applied to speciation studies of blood arsenic species with no need of sample pretreatment. Speciation of arsenic in blood samples collected from two patients after the ingestion of realgar-containing drug reveals slight increase of arsenite and DMA, resulting from the digestion of realgar.     

Determination of anthocyanins and related components in red wines by micro- and capillary HPLC

Anthocyanins and derived components of red wines were determined by microHPLC using a 1 mm ID HPLC column coupled on-line with an MS detector equipped with an ESI (ElectroSpray Ionisation) source. The use of microcolumn HPLC greatly enhanced detection performance, allowing direct identification of components present in the fraction. Nineteen anthocyanins were detected. Fifteen were identified, two were tentatively identified, and only the aglycon of the remaining two components was certainly identified. Six anthocyanin-derived pigments, supposedly formed during wine maturation, were also investigated and found in a wine sample. The analysis of red wine anthocyanins was also carried out by injecting a large sample volume onto a 0.32 mm ID HPLC column, using the column focusing technique, in order to decrease the limit of detection and quantification of components present in a very small amounts.     

Selective and sensitive detection of organic contaminants in water samples by on-line trace enrichment–gas chromatography–mass spectrometry

Liquid chromatographic (LC) type trace enrichment is coupled online with capillary gas chromatography (GC) with mass spectrometric (MS) detection for the analysis of aqueous samples. A volume of 1–10 ml of an aqueous sample is preconcentrated on a trace-enrichment column packed with a polymeric stationary phase. After cleanup with HPLC-grade water the precolumn is dried with nitrogen and subsequently desorbed with ethyl acetate. A fraction of 60 μl is introduced on-line into a diphenyltetramethyldisilazane-deactivated retention gap under partially concurrent solvent evaporation conditions and using an early solvent vapor exit. The analytes are separated and detected by means of GC–MS. The potential of the LC–GC–MS system for monitoring organic pollutants in river and drinking water is studied. Target analysis is carried out with atrazine and simazine as model compounds; the detection limits achieved under full-scan and multiple ion detection conditions are 30 pg and 5 pg, respectively. Identification of unknown compounds (non-target analysis), is demonstrated using a river water sample spiked with 168 pollutants varying in polarity and volatility.     

Determination of delta 1-tetrahydrocannabinol in human fat biopsies from marihuana users by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC/MS) method for analysis of delta 1-tetrahydrocannabinol (delta 1-THC) in human fat samples is described. The fat sample, obtained from heavy marihuana users 1 week before and 4 weeks after smoking, is homogenized in hexane + 2-propanol, centrifuged, and the supernatant mixed with Lipidex 5000. The solvent is evaporated and the dried gel is packed in a glass column. delta 1-THC is eluted from the column with methanol + water + acetic acid, diluted with water and the eluent is passed through a bed of Octadecylsilane-bonded silica. After washing and drying, the retained delta 1-THC is eluted with hexane, derivatized with N-methyl-N-(t-butyl-dimethysilyl)trifluoroacetamide (MTBSTFA) and finally purified by HPLC on an Octadecyl Sl 100 column in methanol. The amount of delta 1-THC is determined by GC/MS, using selected ion monitoring, and a deuterated internal standard. The recovery of delta 1-THC is about 80%, and the concentration of delta 1-THC in the fat samples analysed ranged between 0.4 and 193 ng/g wet tissue.     

On-line HPLC-HRGC coupling and simultaneous transfer of two different LC fractions: Determination of aliphatic alcohols and sterols in olive oil

A modified loop-type interface is decribed which uses two 6-way valves and concurrent eluent evaporation to perform an on-line transfer and simultaneous gas chromatographic analysis of two different fractions pre-separated by liquid chromatography. The interface is used to simultaneously analyze aliphatic alcohols and sterols present in olive oil. LC pre-separation is carried out using a normal phase column (20 cm × 0.21 cm i.d.) and hexane-isopropanol (99:1) as a mobile phase at a flow of 0.2 ml/min; for the GC analysis a 5 % phenyl, 95 % dimethyl siloxane (25 m × 0.32 mm i.d., 0.4 μm film thickness) column is used.     

Automated on-line in-tube solid-phase microextraction coupled with HPLC/MS/MS for the determination of butyrophenone derivatives in human plasma

This paper describes a fully automated on-line method combining in-tube solid-phase microextraction (SPME) in which sample clean-up and enrichment are conducted through an open tubular fused-silica capillary column and high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) detection for the determination of six butyrophenone derivatives (moperone, floropipamide, haloperidol, spiroperidol, bromperidol, and pimozide) in human plasma samples. The six butyrophenones were extracted by repeatedly aspirating and dispensing plasma sample solutions on a DB-17 capillary column (60 cm × 0.32 mm i.d., film thickness 0.25 μm). The analytes retained on the inner surface of the capillary column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. Extraction efficiencies ranged from 12.7% to 31.8% for moperone, spiroperidol, and pimozide, and from 1.08% to 4.86% for floropipamide, haloperidol, and bromperidol. The regression equations for all compounds showed excellent linearity, ranging from 0.05 to 50 ng/0.1 mL of plasma, except for moperone and spiroperidol (0.01 to 50 ng/0.1 mL). The limits of detection and quantification in plasma for each drug were 0.03–0.2 and 0.1–0.5 ng/mL, respectively. The intra- and inter-day coefficients of variation for all compounds in plasma were not greater than 13.7%.     

Organic elemental analysis of small amounts of sample by coulometry: V. Analysis of organic chloropesticides

A combination of a fast current controlled coulometer with a simple gas chromatograph proved to be useful in organo chloropesticide analysis, e.g., in milk. After a clean up, a pesticide containing hexane solution is injected in a simple gas chromatographic column. The excess hexane is exhausted through a special valve and the separated pesticides are combusted successively. Then the combustion products (e.g., HCl) are swept into a coulometric titration cell. The chloride is titrated automatically within 5–10 sec. There was a good agreement between analyses using the described system and analyses carried out using a commercially available gas chromatograph with an electron capture detector. The standard deviation of a series of organo chloropesticide analyses was less than 5% rel.; the recovery was better than 95%. A complete gas Chromatographic analysis after an appropriate clean up takes less than 10 min. The advantages and disadvantages of coulometric detection are noted. A determination of the total organo chloropesticide content using a special flash combustion is possible as well.     

Co-solvent effects for preventing broadening or loss of early eluted peaks when using concurrent solvent evaporation in capillary GC. Part 1: Concept of the technique

The concept and some first results of a method are described for evaporating large volumes of solvent in a relatively short pre-column (retention gap) in such a way that solvent trapping retains volatile components in the inlet up to completion of solvent evaporation. The method was developed for transferring large volumes (easily exceeding 1 ml) of HPLC eluent to GC when using on-line coupled HPLC-GC, but is equally suited for injecting large sample volumes (at least some 50 μl) and could be particularly useful for introducing aqueous solutions. Concurrent solvent evaporation allows introduction of very large volumes of liquid into GC. However, peaks eluted up to some 40–80° above the column temperature during introduction of the liquid are strongly broadened due to the absence of solvent trapping. On the other hand, previous retention gap techniques involving solvent trapping were not suited for transferring very large volumes of liquid into GC. Using a relatively high boiling co-solvent added to the sample or the HPLC eluent, advantages of concurrent solvent evaporation can be combined with solute reconcentration by solvent effects, allowing elution of sharp peaks starting at the column temperature during introduction of the sample.