Coupled LC-GC: Evaporation rates for partially concurrent eluent evaporation using an early solvent vapor exit

Partially concurrent eluent evaporation presupposes an eluent evaporation rate in the GC pre-column that approaches the LC flow rate. Discharging the vapors through the whole GC column, evaporation rates reach 10–30 μl/min, i.e. are suitable just for LC flow rates typical for packed capillary LC columns. With an early vapor exit, evaporation rates are increased to 100–200 μl/min (under extreme conditions to some 800 μl/min), thus fitting the LC flow rates of 2 mm i.d. columns. Evaporation rates were measured for a standard set of pre-columns and conditions. The dependence of the evaporation rate on temperature, inlet pressure, carrier gas, and internal diameter of the retaining pre-column are discussed in order to allow the design of a GC system producing a desired evaporation rate.     

Solvent Trapping during Large Volume Injection with an Early Vapor Exit,Part 2: Chromatographic Results and Conclusions

Solvent trapping reconcentrates volatile components after injection or on-line transfer of large volumes. When an early vapor exit is used, typically after a 5–10 m × 0.53 mm i.d. uncoated precolumn, the solvent trapping process differs from that described previously. The visual experiments and the conclusions drawn therefrom, as described in a previous paper, are supplemented with chromatographic results. They show that even hexane can be quantitatively analyzed in 250 μl of a pentane solution. Injection of a volume of 250 μl by vaporizer/precolumn solvent splitting was used in the analysis of gasoline in drinking water. Conditions for the transfer of a 1000 μl volume can easily be adjusted through detection of the front end of the flooded zone by a thermocouple mounted on the outer wall of the precolumn.     

High temperature vaporizing chambers for large volume GC injections and on-line LC-GC

The suitability of some chambers for sample evaporation at high input flow rates (>100 μl/min) was studied by visual experiments. The chambers were at temperatures far above the solvent boiling point in order to achieve the heat transfer required. Shooting liquid owing to violent evaporation, flooding of the chamber as a result of cooling, and excessively high vapor pressure causing backflow into the gas supply system were found to be the limiting factors. Fused silica capillaries into which a piece of wire or polyimide-free fused silica capillary had been inserted were found to be suitable for the vaporization of “easy” solvents, such as alkanes (up to some 1.7 ml/min), but packed beds were required to achieve favorable evaporation of dichloromethane or methanol/water (up to some 800 and 300 μl/min, respectively).     

Gas chromatographic determination of guanidino compounds in uremic patients using glyoxal as derivatizing reagent

The guanidino compounds guanidine, methylguanidine, guanidinoacetic acid, guanidinopropionic acid, guanidinobutyric acid and guanidinosuccinic acid were eluted and separated after pre-column derivatization with glyoxal from an HP-5 column (30 m × 0.32 mm i.d.) with film thickness 0.25 μm at an initial column temperature of 100 °C for 2 min, with ramping of 20°C/min up to 250 °C and a nitrogen flow rate of 3 mL/min. Detection was by flame ionization detection. Linear calibrations were observed within 0.1-20.0 μmol/L, with limit of detection within 0.024-0.034 μmol/L for each compound. The separation was repeatable with relative standard deviation (RSD) (n = 6) within 1.2-1.8 and 1.1-1.6% in terms of retention time and peak height/peak area, respectively. The method was applied for the determination of the guanidino compounds from serum of uremic patients (n = 7) and healthy volunteers (n = 8), and amounts were observed within 1.33-11.71 and 0.07-0.39 μmol/L with RSD 1.1-3.5 and 1.1-3.0%, respectively. The results were further supported by the standard addition method.     

Analysis of tocopherols in margarine by on-line HPLC–HRGC coupling

Tocopherol analysis in margarine is usually carried out by HPLC after saponification of the sample and extraction of the vitamin compounds; these steps consume both time and solvents. In this paper we propose an on-line HPLC–HRGC coupling method, which allows us to simplify the preparation of the analytical sample. The sample of margarine is solubilized in hexane in an ultrasonic bath in the dark; the filtered solution is then injected into the liquid chromatograph using a normal phase microbore column eluted with hexane–isopropanol 99.8:0.2. The α-tocopherol, which is eluted with some wax esters, is transferred on-line to the gas chromatograph, using a loop-type interface with the concurrent eluent evaporation and solvent vapor exit, thus it is separated from interfering compounds and determined using an Alltech RSL 300 column (22 m × 0.25 μm i.d., 0.2 μm film thickness). The β, γ, and δ–tocopherols are determined in the same LC run, using fluorimetric detection. The analysis was carried out in 50 min.     

Capillary liquid chromatography with UV detection using N,N-diethyl dithiocarbamate for determining platinum-based antitumor drugs in plasma

A capillary liquid chromatography with UV detection (CLC-UV) system has been developed for determining platinum-based antitumor drugs (e.g., cisplatin, carboplatin, and nedaplatin) in plasma based on the pre-column derivatization of platinum with N,N-diethyl dithiocarbamate (DDTC). The chelated platinum separation was carried out on a capillary column (Inertsil ODS-3, 150 mm × 0.3 mm i.d., 3 μm) using an acetonitrile-water mixture (8:2, v/v) as a mobile phase that flowed at 5.0 μL/min. Detection was carried out by absorbance at 254 nm. Chromatographic peak height was found to be linearly related to the spiked concentration of nedaplatin in the blank control plasma from 5.0 ng/mL to 15 μg/mL (r(2)>0.998). The repeatability (n=5) of the chromatographic peak height for 2.5 μg/mL nedaplatin was 2.6% relative standard deviation (R.S.D.). The CLC-UV system, which required only 20 μL of plasma sample, was applied to the determination of total and free form platinum-based antitumor drugs in plasma after injection into rats. The recovery rates (n=5) of total and free form nedaplatin in plasma were 98% and 99%, respectively, and these repeatability were 2.4% R.S.D. and 3.1% R.S.D., respectively. In addition, the recovery rates (n=5) of total and free form carboplatin in plasma were 99% and 99%, respectively, and these repeatability were 2.9% R.S.D. and 0.24% R.S.D., respectively. The concentration-time profiles of total and free form nedaplatin in rat plasma were monitored to determine the pharmacokinetic parameters.     

Capacity of Uncoated 0.53 mm i.d. Pre-Columns for Retaining Sample Liquid in the Presence of a Solvent Vapor Exit

0.53 mm i. d. uncoated precolumns of about 10 m in length followed by a solvent vapor exit have become a standard set-up for large volume on-column injection. It went unnoticed, however, that the introduction of a vapor exit requires two modification of previous working guidelines. First, the capacity of the precolumn to retain sample liquid is increased by a factor of 2.3–3 as a result of the around 100 times higher carrier gas flow rate. Secondly, it must be considered that this gain in retention of liquid is lost again upon closure of the exit: as the gas flow rate is reduced to a few mL/min, the layer of the residual sample liquid expands about 2.3–3 times. Hence, closure should occur late, and a section of the precolumn must be assigned for this secondary spreading.     

Direct determination of endogenous melatonin in human saliva by column-switching semi-microcolumn liquid chromatography/mass spectrometry with on-line analyte enrichment

An analytical method that enables direct and sensitive determination of endogenous melatonin (MLT) in human saliva was developed by means of column-switching semi-microcolumn liquid chromatography (i.d.: 1-2 mm)/mass spectrometry (LC/MS). The system allows direct injection analysis of a 400-microL aliquot of saliva with minimal sample pretreatment (internal standard (IS) addition and vortex mixing) and a relatively short run-time (10 min). The system consists of three columns to attain large volume injection and on-line analyte enrichment. A pre-column packed with a silica-based mixed-functional C8 (4.0 mm i.d. x 20 mm) was used for on-line sample cleanup. MLT and an IS, the d7 isomer of MLT (d7-MLT), were heart-cut by valve switching and enriched at the top of the intermediate trapping column packed with a silica-based C18 (4.0 mm i.d. x 10 mm). Subsequently, the analytes were backflushed into a semi-micro C18 silica column (2.0 mm i.d. x 150 mm) for the final separation. MLT and IS were ascertained by positive electrospray ionization and selected ion monitoring (SIM). MLT was monitored based on its fragment ion at m/z 174.1 by in-source collision-induced dissociation (CID). The validation of this method revealed a detection limit of 2.5 pg mL(-1) at a signal-to-noise (S/N) ratio of 5. The linearity of the method was established in the ranges 5-250 and 100-2500 pg mL(-1) with a coefficient of determination of greater than 0.998. Accuracies, evaluated at five levels in the range 5-1000 pg mL(-1), were between 81 and 108% with a relative standard deviation (RSD) ranging from 1.3-20%. The method was successfully applied for the endogenous saliva MLT monitoring of two healthy subjects.     

对一种分离测定氨基酸方法的改进

 对Waters公司采用 6 氨基喹啉 N 羟基琥珀酰亚胺基 氨基甲酸酯 (AccQ Tag)柱前衍生化测定氨基酸的方法进行了改进。将流动相流速由原来的 1 0mL/min改变为 2 0mL/min ,用AccQ Tag专用柱 (3 9mmi.d .× 15 0mm ,4μm)在 17 5min(原为 35min) (运行周期为 2 2 5min ,原为 45min)内快速分离测定了 18种氨基酸和牛磺酸。用Nova PakC18柱 (3 9mmi.d .× 15 0mm ,4μm) ,Nova PakC18柱 (4 6mmi.d .× 15 0mm ,4μm) ,SymmetryC18柱 (3 9mmi.d .× 15 0mm ,4μm)和WatersXterraRP 18柱等反相C18柱代替AccQ Tag专用柱 ,均可对氨基酸进行快速分离。     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide

A sensitive high-performance liquid chromatographic method with fluorescence detection was developed to determine memantine (MT) in rat plasma. The method consists of pre-column labeling of MT with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and a clean-up step with solid-phase extraction. A good separation of DIB-MT was achieved within 12 min on an octadecylsilica (ODS) column (150 × 4.6 mm i.d.; 5 μm) with a mobile phase of acetonitrile-water (70:30, v/v). The calibration curve prepared with fluoxetine as an internal standard showed good linearity in the range of 10-400 ng/mL (r = .999). The limits of detection and quantitation at signal-to-noise ratios of 3 and 10 were 2.0 and 6.6 ng/mL, respectively. The method was shown to be reliable with precisions of <5% for intra-day and <9% for inter-day as relative standard deviation. The fluorescence property and reaction yield of authentic DIB-MT were also examined. The proposed method was successfully applied to study the pharmacokinetic interaction between MT and methazolamide.     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

Advantages of monolithic over particulate columns for multiresidue analysis of organic pollutants by in-tube solid-phase microextraction coupled to capillary liquid chromatography

The performance of a monolithic C(18) column (150 mm×0.2 mm i.d.) for multiresidue organic pollutants analysis by in-tube solid-phase microextraction (IT-SPME)-capillary liquid chromatography has been studied, and the results have been compared with those obtained using a particulate C(18) column (150 mm×0.5 mm i.d., 5 μm). Chromatographic separation has been carried out under isocratic elution conditions, and for detection and identification of the analytes a UV-diode array detector has been employed. Several compounds of different chemical structure and hydrophobicity have been used as model compounds: simazine, atrazine and terbutylazine (triazines), chlorfenvinphos and chlorpyrifos (organophosphorous), diuron and isoproturon (phenylureas), trifluralin (dinitroaniline) and di(2-ethylhexyl)phthalate. The results obtained revealed that the monolithic column was clearly advantageous in the context of multiresidue organic pollutants analysis for a number of reasons: (i) the selectivity was considerably improved, which is of particular interest for the most polar compounds triazines and phenyl ureas that could not be resolved in the particulate column, (ii) the sensitivity was enhanced, and (iii) the time required for the chromatographic separation was substantially shortened. In this study it is also proved that the mobile-phase flow rates used for separation in the capillary monolithic column are compatible with the in-valve IT-SPME methodology using extractive capillaries of dimensions similar to those used in conventional scale liquid chromatography (LC). On the basis of these results a new method is presented for the assessment of pollutants in waters, which permits the characterization of whole samples (4 mL) in less than 30 min, with limits of detection in the range of 5-50 ng/L.     

An improved LC‐MS/MS method for quantitation of indapamide in whole blood: application for a bioequivalence study

An improved LC‐MS/MS method for the quantitation of indapamide in human whole blood was developed and validated. Indapamide‐d3 was used as internal standard (IS) and liquid–liquid extraction was employed for sample preparation. LC separation was performed on Synergi Polar RP‐column (50 × 4.6 mm i.d.; 4 µm) and mobile phase composed of methanol and 5 mm aqueous ammonium acetate containing 1 mm formic acid (60:40), at flow rate of 1 mL/min. The run time was 3.0 min and the injection volume was 20 μL. Mass spectrometric detection was performed using electrospray ion source in negative ionization mode, using the transitions m/z 364.0 → m/z 188.9 and m/z 367.0 → m/z 188.9 for indapamide and IS, respectively. Calibration curve was constructed over the range 0.25–50 ng/mL. The method was precise and accurate, and provided recovery rates >80% for indapamide and IS. The method was applied to determine blood concentrations of indapamide in a bioequivalence study with two sustained release tablet formulations. The 90% confidence interval for the geometric mean ratios for maximum concentration was 95.78% and for the area under the concentration–time curve it was 97.91%. The tested indapamide tablets (Eurofarma Laboratórios S.A.) were bioequivalent to Natrilix®, according to the rate and extent of absorption. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).     

高效液相色谱-荧光检测法测定罂粟籽和火锅汤料中的罂粟碱

用所建立的高效液相色谱-荧光检测法测定了罂粟籽和火锅汤料中的罂粟碱。采用的色谱柱为RP-C18柱(250 mm×4.6 mm i.d.,5 μm);检测激发波长为285 nm,发射波长为355 nm;流动相为甲醇-0.02 mol/L乙酸铵(体积比为70∶30),流速0.8 mL/min。实验结果表明,罂粟碱的进样量为1×10-4~0.1 μg时其质量浓度与相应峰面积有良好的线性关系,最低检测限(以信噪比大于3计)达到0.02 ng。罂粟籽中罂粟碱的回收率为99.0%～100.8%。方法快速准确,简便灵敏,分离度高,能够满足有关食品中罂粟碱的检测要求。     

Determination of organochlorine compounds in fatty matrices: Application of normal-phase LC clean-up coupled on-line to GC/ECD

Off-line normal-phase LC has been used for the clean-up of compounds in our laboratory for several years. On-line coupling of this LC system, which typically yields 12 ml fractions, is not possible due to its large fraction volume. The maximum transfer volume in on-line LC-GC/ECD is approx. 300 μl. Therefore down-scaling of the LC system was attempted in order to reach these low fraction volumes. Miniaturization resulted in a 240 μ1 LC fraction containing the analytes of interest, which is transferred to GC/ECD via an on-column interface. Sensitivity requires that a minimum amount equivalent to 1–2 mg of sample should reach the GC detector; the selectivity is determined by the separation between the matrix and the last eluting target analyte. For the analysis of fatty samples, limitations were observed in the separation of dieldrin from triglycerides. Other organochlorine compounds, e.g. polychlorobiphenyls (PCBs), the DDT group, HCB and the HCHs can be analyzed with RSDs of 2–4 % (n = 10) at concentration levels of sub-μ/kg in milk fat using a 3 μm Hypersil silica 50 × 1.0 mm i.d. LC column.     

Solvent Trapping during Large Volume Injection with an Early Vapor Exit. Part 3: The Main Cause of Volatile Component Loss during Partially Concurrent Evaporation

When 0.53 mm i.d. uncoated precolumns connected to a solvent vapor exit are used for sample introduction with partially concurrent solvent evaporation, substantial losses of volatile solutes are often observed. They were found to be the consequence of solute accumulation at the front end of the flooded zone, which in turn is the result of a strong pressure drop over the flooded zone owing to the formation of plugs of sample liquid. The pressure drop causes significant solvent evaporation at the front, which enriches the solute material there and causes its loss. The use of 0.32 mm i.d. restrictions between the uncoated precolumn and the vapor exit greatly reduced this problem.     

Determination of serum estradiol by normal-phase high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

Estradiol extracted with an ODS minicolumn from serum (500 μl) is derivatized with 5-dimethylamino-1-naphthalenesulfonyl (dansyl) chloride at room temperature for 80 min, purified on the ODS minicolumn, separated on silica gel columns (150 × 1.9 mm i.d. and 100 × 1.9 mm i.d.) with n-hexane/chloroform/ethanol (70:30:0.1) as eluent at 3 ml min^?1 and detected by the chemiluminescence emission produced in a post-column reaction with bis-(2,4,6-trichlorophenyl) oxalate (8 mM in chloroform containing 100 mM triethylamine) and hydrogen peroxide (600 mM in methanol containing 6% (v/v) sodium acetate buffer at pH 4.0). The overall recovery of estradiol from serum is ca. 90% and the detection limit is ca. 50 pg.