Quantitative liquid chromatography-mass spectrometry determination of catechins in human plasma by automated on-line extraction using turbulent flow chromatography

A simple, fast and sensitive liquid chromatography-mass spectrometry (LC-MS) method with automated on-line extraction using turbulent flow chromatography (TFC) for the determination of five catechins in human plasma was developed. In this method, after on-line extraction by its injection onto an extractor column at turbulent flow, five catechins were backwashed onto a reversed phase column via on-line column switching and separated chromatographically at a laminar flow of 1 ml min(-1). Using this tandem LC-LC-MS system, the extraction, the separation and the quantitation of five catechins in human plasma could be achieved with satisfactory selectivity and sensitivity. The limit of detection (S/N = 3) ranged from 0.6 to 2 ng ml(-1). The described procedure was very simple and rapid since no off-line sample preparation was required, total analysis time being 18.5 min.     

Analysis of 1-nitropyrene in air particulate matter standard reference materials by using two-dimensional high performance liquid chromatography with online reduction and tandem mass spectrometry detection

1-Nitropyrene (1-NP) is enriched in diesel exhaust particulate matter (DPM) relative to other sources of particulate matter (PM), and has been proposed as a marker for DPM. However, in ambient air, 1-NP concentrations are typically in the low pg/m(3) range. Therefore, collection of large volume air samples coupled with extensive sample clean-up procedures has been required to achieve adequate detection limits to measure 1-NP in ambient samples. We report here an improved LC-MS/MS method suitable for the detection and quantification of 1-NP in low volume ambient PM samples. The method involves ultrasonic extraction of ambient PM in organic solvent, concentration of the sample under reduced pressure, and two-dimensional HPLC analysis of the extract. 1-NP is isolated on the first HPLC column, then converted to 1-aminopyrene (1-AP) via online reduction in a column packed with a Pt/Rh catalyst. The 1-AP containing fraction from the first column is refocused on a trapping column, then eluted through a second HPLC column prior to MS/MS detection. Deuterated (d(9)) 1-NP (1-dNP) is added to each sample prior to extraction as an internal standard for quantification of 1-NP. The accuracy and precision of the assay, as applied to ambient particulate standard reference materials are 110+/-5.7% for SRM 1650b, 116+/-7.1% for SRM 2975, 108+/-5.8% for SRM 1649a, and 53+/-9.2% for SRM 1648. The analytical limit of detection was 152 fg on column, and analytical limit of quantitation 221 fg on column. To our knowledge, the sensitivity of this method is comparable with GC-NICI-MS methods while having the advantage of considerably less extensive sample preparation. This method is an approximately 10-fold improvement in sensitivity over HPLC methods utilizing fluorescence and/or chemiluminescence detection.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Enantioselective analysis of (<Emphasis Type="Italic">R</Emphasis>)- and (<Emphasis Type="Italic">S</Emphasis>)-carvedilol in human plasma by high-performance liquid chromatography

Summary An HPLC column-switching method has been developed and validated for the enantioselective determination of (R)- and (S)-carvedilol in human plasma. Sample preparation was performed either off-line, by extraction with trichloromethane and back-extraction into 0.01m aqueous citric acid which was injected on to a LiChrosorb RP 8 column, or on-line, by injecting diluted (0.1m formic acid) plasma on to a LiChrosorb ADS column. In both instances separation was performed by gradient elution and on-line transfer of the fraction containing, the carvedilol on to an enantioselective Teicoplanin column. The enantiomers of carvedilol were separated isocratically by use of methanol-acetonitrile-triethylammonium acetate, 70:30:0.05 (v/v/w), as mobile phase. With fluorescence detection the limits of quantitation were 0.30 ng mL⁻¹ for (R)-carvedilol and 0.26 ng mL⁻¹ for (S)-carvedilol; these were sufficient to enable investigation of the effect of exercise on plasma concentrations of (R)- and (S)-carvedilol after oral administration of either the racemate or the pure enantiomers. Although the operating conditions were optimized for sample preparation by on-line deproteination on a LiChrospher RP 18 ADS column, the complete method was insufficiently rugged for analysis of large numbers of plasma samples—the enantioselectivity of the Teicoplanin column deteriorated too rapidly because of the transfer of enantioselectivity-poisoning interferences which could not be suppressed sufficiently. In contrast the liquid-liquid sample-extraction procedure combined with column switching resulted in a analytical method with long-term stability. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Very sensitive differential pulse adsorptive stripping voltammetric determination of 4-nitrophenol at poly (diphenylamine)/multi-walled carbon nanotube-β-cyclodextrin-modified glassy carbon electrode

In this work, a glassy carbon electrode (GCE) modified with poly (diphenylamine)/multi-walled carbon nanotubes-β-cyclodextrin (PDPA/MWCNT-β-CD) film was constructed and used for the determination of 4-nitrophenol (4-NP). Diphenylamine was successfully electropolymerised onto MWCNT-β-CD-modified GCE by cyclic voltammetry in monomer solution and 5 mol L^?1 H₂SO₄. The surface morphology of PDPA/MWCNT-β-CD film was characterised using scanning electron microscopy and electrochemical impedance spectroscopy. After adsorption of 4-NP on PDPA/MWCNT-β-CD at 0.2 V for 150 s, it showed a well-defined reduction peak in phosphate buffer solution at pH = 7. The PDPA/MWCNT-β-CD film enhanced the reduction peak current due to the complex formation between β-CD and 4-NP, presence of conductive polymer film as electron transfer mediator and also ability of MWCNTs for strong adsorptive and catalytic effect. Peak current increased linearly with 4-NP concentration in the range of 0.1 to 13.9 µg L^?1. The detection limit was obtained as 0.02 µg L^?1, which is better than other reported detection limits for the determination of 4-NP. The results showed that modified electrode has good sensitivity and selectivity. This sensor was used for the determination of 4-NP in water samples.     

Direct determination of non-steroidal anti-inflammatory drugs by column-switching LC-MS

A method for determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma samples without time-consuming sample pre-treatments was developed. The system consisted of two pumps for mobile phase delivery, a six-port switching valve, a pre-column (Oasis HLB Cartridge Column), and a reversed phase analytical column (COSMOSIL 3C18-MS-II). The analytes were trapped on the precolumn and subsequently separated on the analytical column. The present method allowed on-line sample clean-up and enrichment, leading to improved sensitivity without any tedious sample preparation. The recoveries of NSAIDs from human plasma by column-switching were greater than 72.6%. The total analysis time for a single analytical run was approximately 11 min. The detection limits of NSAIDs were 0.0025 to 0.2 microg/mL using the selected ion monitoring mode.     

Direct enantiomeric analysis of amphetamine in plasma by simultaneous solid phase extraction and chiral derivatization

Summary An analytical approach has been developed for the one step determination of enantiomeric amphetamine composition in plasma, using on-line, pre-column solid phase derivatization with reversed phase HPLC separation. The high molecular weight protein components were excluded by the small pore structure of the polymer and washed out of the reaction column before derivatization. Spiked amphetamine in human plasma was extracted and derivatized by the polystyrene based FMOC-L-prolyl solid phase reagent. The derivatized diastereomers were separated on a conventional ODS column with an ACN/H₂O mobile phase. No kinetic resolution or racemization was observed in this solid phase derivatization. Calibration plots and reproducibility experiments were performed to demonstrate the validity of the new approach. Automation of the procedure provided a simple and reproducible method for direct chiral recognition in plasma samples.     

Determination of grepafloxacin in plasma samples by HPLC: Application to clinical pharmacokinetic studies

Summary A sensitive HPLC assay for the determination of grepafloxacin (GRE) in biological samples is described. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1M), followed by extraction with trichloromethane. GRE and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column using an aqueous phosphate solution-acetonitrile (78∶22) mobile phase. The concentrations of ENR and GRE eluting of the column with retention times of 2.55, and 4.90 min, respectively were monitored by fluorescence atλ _ex 338 andλ _em 425 nm. The method was shown to be linear from 5 to 4000 ng mL⁻¹. The detection and quantitation limits were 5 and 10 ng mL⁻¹, respectively. Mean recovery was determined as 90%. Inter- and intra-assay precisions were 3.0% and 3.5% respectively. The method was applied to the determination of GRE in plasma samples collected during clinical pharmacokinetic studies.     

Determination of tacrolimus (FK 506) in whole blood using liquid chromatography and fluorescence detection

Summary A sensitive and selective liquid chromatographic method, using precolumn derivatization with dansylhydrazine followed by fluorescence detection, has been developed for the determination of tacrolimus (FK 506) in whole blood. After haemolysis, whole blood samples are extracted with diethyl ether and derivatized. After on-line removal of excess dansylhydrazine on a C₁₈ precolumn, the derivative is loaded on to a C₁₈ clean-up column, and a heart cut is subsequently transferred to a graphitized carbon column, where the final separation takes place. The method requires 1 ml of sample and has a limit of quantitation of 3 ng/ml. At the 15 ng/ml level the precision isca 10%, and the response is linear from the limit of quantitation toca 200 ng/ml of FK 506 in whole blood. The capacity of the method is 50 samples/day and about 1000 1-ml samples can be analyzed without changing either clean-up or separation column. Finally, the applicability of the method for therapeutic drug monitoring is shown.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

在线富集-HPLC法测定水溶液中多环芳烃

本文采用在线富集-HPLC 法测定水溶液中多环芳烃。先使样品中的组分浓缩在一支短的富集柱上,然后切换阀门,将富集物洗脱到 RPHPLC 的体系中进行分离,并用紫外和荧光检定器对冼脱物进行检测。为了提高检测灵敏度和选择性,实验中采用了荧光波长程序技术。考察了富集的最佳条件,并比较了在线法与离线法的测定结果。     

Quantification of trovafloxacin in serum by high-performance liquid chromatography with on-line solid-phase extraction

Summary A rapid, simple, accurate and sensitive liquid chromatographic assay with on-line solid-phase extraction is described for determination of trovafloxacin in human serum. Samples were deproteinized with acetonitrile and injected on to an NH₂ extraction column for sample clean-up. Thereafter, an on-line column-switching system was used for quantitative transfer of the drug to a C₁₈ analytical column. Separation was performed by ion-pair chromatography and detection was by ultraviolet absorbance at 275 nm. Recovery was 98.5%. The linear range was from 0.25 to 20μg mL⁻¹, with a correlation coefficient of 0.999. Detection limit was 0.1 μg mL⁻¹ from extraction of 25 μL serum.     

Trace analysis of surfactants derived from fatty alcohols I. Optimization of derivatization reaction

Summary Pre-column derivatization of the primary hydroxyl group in fatty alcohols and fatty alcohol ethoxylates using carbazole-9-carbonyl chloride (CC−Cl) and FMOC-Cl is described and compared with derivatization with 1-naphthoyl chloride (N−Cl). As the excess of derivatization reagent leads to a broad and strongly tailing reagent peak, it hinders trace determination of fatty alcohols and fatty alcohol ethoxylates. Therefore, an off-line as well as an on-line solid-phase extraction (SPE) method for removal of excess reagent are described. The on-line method which is based on column switching, shows better reproducibility higher pre-concentration, lower risk of contamination and can be easily automated, while the off-line method is better suited for the analysis of derivatized, fatty alcohol ethoxylates. An example of the trace analysis of fatty alcohols with a concentration of 2 ppb is given.     

Sensitive and selective determination of 4-nitrophenol in water and food using modified polyethyleneimine-capped carbon dots

Hazardous 4-nitrophenol (4-NP) has created serious threats to humans and the environment; therefore, it is highly desirable to develop a facile and practical method for the monitoring of 4-NP in environment and food. Here, a fluorescence method based on modified polyethyleneimine-capped carbon dots (mPEI-CDs) was developed for sensitive and selective determination of 4-NP in water, fruit, and vegetable samples. First, highly fluorescent mPEI-CDs (quantum yield about 40.3%) were easily synthesized via a one-step hydrothermal method by using novel acetic anhydride modified polyethyleneimine (mPEI) and citric acid as precursors. Compared to the unmodified PEI-CDs, the acetic anhydride mPEI-CDs exhibited excellent fluorescent stability in a wider pH range of 4.0–9.0. Under pH 8.0, a selective determination of 4-NP was achieved based on the inner filter effect (IFE) mechanism. After optimization, good linear relationships between fluorescence intensity function (F₀-F)/F₀ and the concentration of 4-NP were obtained in ranges of 0.5–10 and 10–100 μM, respectively, while efficiently avoiding the interferences from two other nitrophenol isomers, possible coexisting metal cations and anions in samples. Finally, the proposed approach was successfully applied for the determination of 4-NP in water, honey, strawberry, and tomato samples.     

Fast Determination of Sudan I-IV in Chili Products Using Automated On-Line Solid Phase Extraction Coupled with Liquid Chromatography-Mass Spectrometry

A rapid, accurate and precise method for the determination of sudan I-IV in chili products using on-line solid phase extraction and LC-MS has been developed. Chili products were extracted with acetone and the analytes were cleaned up and enriched on an SPE column (C₁₈, 15–40 µm) through on-line SPE. Chromatographic separation was performed on a C₁₈ analytical column (2.1 × 150 mm, 3 µm) with gradient elution programming of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. All four sudan dyes were separated in less than 8 min. Using in-house validation data, linearity coefficients of determination (R²) of more than 0.9997 were obtained. The limits of detection (LOD) and limits of quantitation (LOQ) for sudan I, II and IV were 0.03 and 0.05 mg kg^?1, respectively, and 0.04 and 0.1 mg kg^?1 for sudan III. The intra- and inter-day recoveries of the four sudan dyes in chili powder were between 90.1–101.6% and 90.2–102.0%, respectively, with relative standard deviation (RSD) between 0.014–0.164% and 0.011–0.202%, respectively. Therefore, this proposed method could be an alternative assay for the determination of sudan I-IV in chili products due to its rapidness, sensitivity, less sample and solvent consumption.     

Novel on‐line column extraction apparatus coupled with binary peak focusing for high‐performance liquid chromatography determination of rifampicin in human plasma: A strategy for therapeutic drug monitoring

In order to develop a method that is completely suitable for the routine therapeutic drug monitoring, a sensitive and fully automated on‐line column extraction apparatus in combination with high‐performance liquid chromatography allowing binary peak focusing was developed and validated for the determination of rifampicin in human plasma. Rifapentine was used as an internal standard. The analytical cycle started with the injection of 100 μL of the sample pretreated by protein precipitation in a Venusil SCX extraction column. After the elution, the analytes were transferred and concentrated in an Xtimate C₁₈ trap column. Finally, the trapped analytes were separated by an Xtimate C₁₈ analytical column and were analyzed by an ultraviolet detector at 336 nm. With this new strategy, continuous on‐line analysis of the compounds was successfully performed. The method showed excellent performance for the analysis of rifampicin in plasma samples, including calibration curve linearity (All r were larger than 0.9996), sensitivity (lowest limit of quantification was 0.12 μg/mL), method accuracy (within 6.6% in terms of relative error), and precision (relative standard deviations of intra‐ and interday precision were less than 7.8%). These results demonstrated that the simple, reliable, and automatic method based on on‐line column extraction and binary peak focusing is a promising approach for therapeutic drug monitoring in complex biomatrix samples.     

Determination of perfluorooctane sulfonate in river water by liquid chromatography/atmospheric pressure photoionization mass spectrometry by automated on-line extraction using turbulent flow chromatography

A simple, fast and sensitive liquid chromatography/atmospheric pressure photoionization mass spectrometry (LC/APPI-MS) method, with automated on-line extraction using turbulent flow chromatography (TFC), was developed for the determination of perfluorooctane sulfonate (PFOS) in river water. In this method, following an on-line extraction by injection onto a column under TFC conditions, PFOS is back-flushed onto a reversed-phase column via on-line column switching, and resolved chromatographically at a laminar flow rate of 1 mL min(-1). Using this tandem LC-LC/APPI-MS system the extraction, separation and selective detection of PFOS in river water could be achieved with satisfactory selectivity and sensitivity. The limit of detection (LOD) (S/N = 3) and the limit of quantitation (LOQ) (S/N = 10)were 5.35 and 17.86 pg mL(-1). The described procedure was very simple since no off-line sample preparation was required, total analysis time being 18.75 min.     

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.     

Analysis of ofloxacin in plasma samples by high-performance liquid chromatography

Summary A sensitive HPLC method has been developed for determination of ofloxacin (OFL) in biological fluids. Sample preparation was performed by adding phosphate buffer (pH 7.4, 0.1m) then extraction with trichloromethane. OFL and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column with aqueous phosphate solution-acetonitrile, 80∶20, as mobile phase. The fluorescence of the column effluent was monitored at λ_ex 338 and λ_em 425 nm. The retention times were 2.66 and 4.24 min for OFL and SAR, respectively, and the detection and quantitation limits were 8 and 15 ng mL⁻¹, respectively. Plots of response against ofloxacin concentration were linear in the range 8 to 2000 ng mL⁻¹. Recovery was 92.9% for OFL.     

On-line deproteinization of serum sample for HPLC analysis of hydrophilic compounds and its application to gentamicin

Summary The binding of serum proteins with Butyl Toyopearl (BT) 650-M has been investigated and applied to on-line deproteinization for the HPLC determination of gentamicin components, c₁, c_1a, c₂, in serum. It was found that in 0.4% perchloric acid medium about 36mg of BSA was adsorbed on 1ml of wet gel. Under this condition hydrophilic components such as gentamicin passed through the pre-column packed with BT 650-M, while serum proteins and hydrophobic components were trapped in the pre-column. The ion pair between gentamicin components and pantanesulfonate anion was effectively trapped in a reversed-phase analytical column. It was then eluted and fluorometrically determined by post-column derivatization with o-phthalaldehyde. The recovery was quantitative with good reproducibility at therapeutic concentrations in sera. Several clinical samples were analyzed by the method.