Generation of picoliter droplets of liquid for electrospray ionization with piezoelectric inkjet

We report the association of inkjet and electrospray ionization MS to detect picoliter droplet, where the liquid volume and its position onto the tip can be precisely controlled to form ultrafine droplets for successive ionization of the analyte. Single rectangle pulse was applied to piezoelectric device on inkjet microchip for the ejection of each picoliter droplet, and it was controlled by a computer. The voltage and width of driving pulse for the inkjet were optimized to make reproducible ejection of the solvent with low viscosity. The volume of each droplet was about 600 pl, and a trigger of 10 droplets was selected as the best inlet mode taking relative standard derivation of the droplets into consideration. The target substrate used with high voltage to form ionization was graphite, after several attempts with some materials. High‐speed camera was used to capture the breaking‐up process of a droplet. The distance between the inkjet nozzle and the tip was set at 2 cm to avoid short circuit. The influences on the mass intensity of the diameter of the tip, the volume and the concentration of the sample were examined. The tip with a small diameter performed greater intensity, and the limit of detection decreased, whereas the small volume of liquid played high ionization efficiency. Linear regression in the range between 1 and 200 ppm for caffeine was conducted, where internal standard theobromine was used. Some real samples were also detected with the instrument. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of substituted purines in body fluids by micellar electrokinetic capillary chromatography with direct sample injection.

Many substituted purines (theobromine, caffeine, paraxanthine, theophylline and uric acid, as well as other methylated xanthines and uric acids) can easily be separated and analysed in one run using micellar electrokinetic capillary chromatography with a boratephosphate buffer containing 75 mM sodium dodecyl sulphate (pH approximately 9). Serum, saliva and urine samples collected after the self-administration of caffeine and serum samples from patients receiving theophylline or caffeine pharmacotherapy were screened for substituted purines. The data presented show the ease of using on-column multi-wavelength detection for investigating the feasibility of direct sample application, the characterization of sample pretreatment procedures and peak confirmation by comparing absorption spectra. It is shown that the determination of purines in serum and saliva samples, including therapeutic concentrations of caffeine and theophylline, can be accomplished without any sample pretreatment, whereas sample extraction is required for the determination of purines in urine. Quantitative data for the determination of micromolar amounts of theophylline (samples from adult patients) and caffeine (samples from infants born prematurely) in serum samples compared well with data obtained by non-isotopic immunoassays. Micellar electrokinetic capillary chromatography with the direct injection of serum or saliva samples requires only microlitre volumes of sample and several different compounds can be determined within a few minutes.     

Quantitative capillary electrophoresis and its application in analysis of alkaloids in tea,coffee, coca cola,and theophylline tablets

A quantitative CE (qCE) system with high precision has been developed, in which a 4‐port nano‐valve was isolated from the electric field and served as sample injector. The accurate amount of sample was introduced into the CE system with high reproducibility. Based on this system, consecutive injections and separations were performed without voltage interruption. Reproducibilities in terms of RSD lower than 0.8% for retention time and 1.7% for peak area were achieved. The effectiveness of the system was demonstrated by the quantitative analysis of caffeine, theobromine, and theophylline in real samples, such as tea leaf, roasted coffee, coca cola, and theophylline tablets.     

统计模拟分光光度法同时测定复方茶碱片中的五组分含量

研究了测定复方茶碱片中五组分含量的统计模拟分光光度法。利用逐步回归分析法建立经验数学模型。再运用改良单纯形法进行优化，同时测定复方茶碱片中的五种组分-氨基比林、非那西丁、茶碱、可可碱和咖啡因的含量，其回收率分别为99.5%、100.2%、101.2%、100.2%和99.2%，RSD分别为1.51%、1.03%、4.44%、2.47%和3.12%。t检验结果证明，该方法与HPLC法之间无显著性差异。     

Simultaneous determination of methylxanthines in coffees and teas by UV-Vis spectrophotometry and partial least squares

This work describes a method to simultaneously determine caffeine (CF) and theobromine (TB) in coffee and tea samples using partial least squares (PLS-1). Sample preparation was required to eliminate strong interfering components. High-performance liquid chromatography (HPLC)-found concentrations of caffeine and theobromine (theophylline was not found in any analyzed sample) were used to construct universal calibration matrixes for coffee and tea. Due to the low levels of theobromine when compared to caffeine (up to 1000:1), theobromine addition standard was required to dramatically improve method performance. The method developed did not show statistically significant differences with an HPLC standard technique.     

Measurement of caffeine and its three primary metabolites in human plasma by HPLC‐ESI‐MS/MS and clinical application

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high‐performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC‐ESI‐MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects.     

Simultaneous separation and analysis of camptothecin alkaloids in real samples by large‐volume sample stacking in capillary electrophoresis

Large‐volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether‐β‐cyclodextrin and 20 mm ionic liquid 1‐ethyl‐3‐methyllimidazole l ‐lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 μg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.     

Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography

This work relates the development of an analytical methodology to simultaneously determine three methylxanthines (caffeine, theobromine, and theophylline) in beverages and urine samples based on reversed-phase high-performance liquid chromatography. Separation is made with a Bondesil C18 column using methanol-water-acetic acid or ethanol-water-acetic acid (20:75:5, v/v/v) as the mobile phase at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for all three methylxanthines. This method is applied to urine and to 25 different beverage samples, which included coffee, tea, chocolate, and coconut water. The concentration ranges determined in the beverages and urine are: < 0.1 pg/mL to 350 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/mL for theophylline. The method proposed in this study is rapid and suitable for the simultaneous quantitation of methylxanthines in beverages and human urine samples and requires no extraction step or derivatization.     

Caffeine and theobromine in coffee, tea and cola-beverages

Summary A new method based on reversed-phase ion-interaction HPLC is described for the identification and quantitation of theobromine, theophylline and caffeine in beverages. The interaction reagent used is octylamine orto-phosphate which also constitutes the mobile phase. The stationary phase is a microparticulate reversed-phase C-18 packing. With spectrophotometric detection at 274 nm, detection levels of 0.15, 0.30 and 0.40 ppm were achieved for theobromine, theophylline and caffeine, respectively.Quantitative analysis was performed by the standard addition method for theobromine and caffeine contents in tea, espresso-coffee, decaffeinated coffee, decaffeinated tea and cola-beverages.     

Determination of chlorophenols in water using dispersive liquid–liquid microextraction coupled with water‐in‐oil microemulsion electrokinetic chromatography in normal stacking mode

The current routes to couple dispersive liquid–liquid microextraction with capillary electrophoresis are the evaporation of water immiscible extractants and the back‐extraction of analytes. In this study, a new methodology for this combination using water‐in‐oil microemulsion electrokinetic chromatography coupled with normal stacking mode on‐line sample concentration was developed to analyze chlorophenols in water samples. The analytes were extracted with tributyl phosphate and the extractant dilution (3×) was directly injected into an electrophoresis buffer (7.7 cm) containing 5% sodium dodecyl sulfate, 78% 1‐butanol, 2% 1‐heptane, and 15% sodium acetate solution (pH 8.0). This proposed method is very simple and convenient compared to the conventional procedures. The key parameters affecting separation and concentration were systematically optimized. Under the optimized conditions, dispersive liquid–liquid microextraction contributed an enrichment factor of 45–50, and the overall sensitivity improvement was 312–418‐fold. Limits of detection between 1.4 and 3.0 ng/mL and limits of quantification between 4.5 and 10.2 ng/mL were achieved. Acceptable repeatability lower than 3.0% for migration time and 9.0% for peak areas were obtained. The developed method was successfully applied for analysis of the chlorophenols in real water samples.     

Simultaneous enrichment and separation of neutral and anionic analytes through combining large volume sample stacking with sweeping in CE

In this work, we overcame the deficiencies of large volume sample stacking (LVSS) in separating low‐mobility and neutral analytes through combining LVSS with sweeping in CE, and employed this new approach to enrich and separate neutral and anionic analytes simultaneously. This technique was carried out with pressure injection of large‐volume sample followed by EOF as a pump pushing the bulk of low‐conductivity sample matrix out of the outlet of the capillary while analytes were swept by micelles and separated via MEKC without the electrode polarity switching. Careful optimization of the enrichment and separation conditions allowed the enrichment factors (EFs) of peak height and peak area of the analytes to be in the range of 9–33 and 21–35 comparing with the conventional injection mode, respectively. The five analytes were baseline separated in 15 min and the detection limits ranged from 26.5 to 55.8 ng/mL (S/N = 3). The developed method was successfully applied to determine adenine, caffeine, theophylline, reduced L‐glutathione (GSH) and oxidized L‐glutathione (GSSG) in two different teas with recoveries that ranged from 84.4 to 105.2%.     

Poly(aquachlorobis(1,10–phenanthroline)copper(II)iodidemonohydrate)/GCE for simultaneous determination of caffeine and theophylline in human serum,tea, and tablet samples

This paper presents metal complex based polymer film modified electrode for simultaneous determination of caffeine, and theophylline. Potentiodynamic fabrication of poly(aquachlorobis(1,10– phenanthroline)copper(II)iodidemonohydrate) modified glassy carbon electrode (poly(ACP₂CuIH)/GCE) was verified using cyclic voltammetric and electrochemical impedance spectroscopic techniques. In contrast to the unmodified glassy carbon electrode, the poly(ACP₂CuIH)/GCE in equi-molar mixture of theophylline and caffeine revealed sufficiently separated oxidative peaks with much enhanced peak current showing electrocatalytic property of the polymer film towards the oxidation of theophylline and caffeine. Under optimized solution pH and square wave voltammetric parameters, oxidative peak current response of poly(ACP₂CuIH)/GCE showed linear dependence on the concentration of caffeine and theophylline in the concentration range 1.0–200.0 µM with limit of detection 8.92 × 10^-3 µM for theophylline, and 1.02 × 10^{-2 µ}M for caffeine. Spike recovery in the range 97.0-102.4% for theophylline, and 95.4-100.0% for caffeine, interference recovery in the range 96.0–101.0% for theophylline, and 95.7–104.3% for caffeine, agreement of the detected amounts of theophylline and caffeine in tablet samples with the nominal values, and stability of the modified electrode all validated the developed method for simultaneous determination of theophylline and caffeine in wide range of real samples. The method was applied for simultaneous determination of both theophylline and caffeine in three tea brands (Black lion, Addis, and Wush wush), pharmaceutical tablet brands (Panadol extra, and Theodrine), and human blood serum samples making the method an excellent candidate.     

Coulometric Determination of Purine Alkaloid Series with Electrogenerated Chlorine

In acid solution, caffeine, theobromine, and theophylline are rapidly and quantitatively oxidized with electrogenerated chlorine in a stoichiometric ratio of 2 : 5 to give alloxan and urea. Procedures for the coulometric determination of microgram amounts of caffeine, theophylline, and theobromine in model solutions, caffeine in tea and coffee samples, and caffeine and theophylline in some pharmaceutical preparations were developed.     

Zirconia based monoliths used in hydrophilic-interaction chromatography for original selectivity of xanthines

Monolithic capillary columns based on zirconia were prepared directly from zirconium alkoxide. They were also prepared coating a classical silica based monolithic column with zirconium butoxide. Using the gradual evolution of the theophylline/caffeine separation factor, it was found that successive zirconia coatings produced the progressive fading of surface silanols replaced by Zr–OH groups. The behavior of a silica monolith coated four times with zirconium butoxide was very similar to that of a pure zirconia monolith. The dramatic change in xanthine separation factor observed with zirconia stationary phases and the theophylline and caffeine probe solutes was used to develop a complete separation of xanthines on zirconia stationary phase in less than 6 min. The three dimethylxanthine isomers, theophylline, theobromine and paraxanthine, are very difficult to separate in RPLC with classical C₁₈ stationary phases. The three isomers were easily separated in HILIC mode on a zirconia based stationary phase.     

Bestimmung von Coffein,Theobromin und Theophyllin in Tee,Kaffee, Kakao und Getränken durch Hochdruckflüssigkeitschromatographie mit elektrochemischem Detektor

Zusammenfassung Eine Methode zur quantitativen Bestimmung von Coffein, Theobromin und Theophyllin wurde ausgearbeitet. Nach Untersuchung des voltammetrischen Verhaltens dieser Verbindungen an einer glasartigen Kohlenstoffelektrode zeigte sich, daß eine gleichzeitige voltammetrische Bestimmung infolge der ähnlichen Halb-Peakpotentiale nicht möglich ist. Daher wurden die Methylxanthine durch Hochdruckflüssigkeitschromatographie getrennt und mit einem amperometrischen Detektor angezeigt.Angewandt wurde die Methode zur Bestimmung des Coffeins, Theobromins und des Theophyllins in Tee, Kaffee, Kakao und Getränken. Nach Extraktion mit Wasser wurden störende Substanzen an Polyamid gebunden und die Methylxanthine durch reversed phase chromatography getrennt. Die Identifizierung und die quantitative Bestimmung erfolgte mit dem elektrochemischen Detektor. Die Nachweisgrenze liegt für Coffein bei 4 ng, für Theobromin bei 1,5 ng und für Theophyllin bei 0,7 ng.

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Determination of caffeine, theobromine and theophylline in tea, coffee, cocoa and beverages by high fressure liquid chromatography with electrochemical detection

Summary A method for quantitative determination of caffeine, theobromine and theophylline was developed. By investigation of the voltammetric behaviour of these substances at a glassy carbon electrode it was found, that a simultaneous voltammetric determination is impossible because the half-peak potentials of theobromine and caffeine are nearly identical. Therefore the methylxanthines were separated by HPLC and detected with an amperometric detector.This method was applied to the determination of caffeine, theobromine and theophylline in tea, coffee, cocoa and in beverages. After extraction with water interfering substances were removed on a polyamide column and the extract was separated by reversed phase chromatography. For identification and quantitative determination the electrochemical detector was used. The detection limit for caffeine is 4 ng, for theobromine 1,5 ng and for theophylline 0,7 ng.

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Vorgetragen beim 8. Internationalen Mikrochemischen Symposium in Graz, 28. August 1980.     

On‐line synergistic stacking in capillary zone electrophoresis featuring field‐amplified sample stacking and micelle to cyclodextrin stacking in the determination of two alkaloids in complicated matrix samples

A novel on‐line synergistic proconcentration strategy coupling field‐amplified sample stacking and micelle to cyclodextrin stacking for cationic analytes in capillary zone electrophoresis has been proposed and applied for the separation and determination of two alkaloids, matrine, and oxymatrine in complicated matrix samples. The approach was performed by the long injection of sample in a low‐conductivity sodium dodecyl benzene sulfonate solution followed by the injection of hydroxypropyl‐β‐cyclodextrin solution in higher conductivity. The stacking mechanism of this method has been expounded and parameters affecting stacking effect have been optimized in our study. Under the optimum experimental conditions, 169‐ and 218‐fold sensitivity improvements were achieved for matrine and oxymatrine when compared with normal injection. Analytical indicators including linearity, limits of detection, and reproducibility (intra‐ and inter‐day relative standard deviations) were evaluated. Moreover, sample matrix effect was studied using compound flavescent sophora and salicylic acid powder and spiked urine samples. The developed method is an attempt for the combination of micelle to cyclodextrin stacking with other stacking methods. It could be a good alternative choice for the determination of alkaloids in a complex sample matrix.     

Determination of caffeine and caffeine-related metabolites in ephedra-containing standard reference materials using liquid chromatography with absorbance detection and tandem mass spectrometry

The concentrations of caffeine and caffeine-related compounds in 2 ephedra-containing reference materials have been determined by 3 independent methods with measurements performed by the National Institute of Standards and Technology (NIST) and a collaborating laboratory. Results from the 3 methods were used for value assignment of caffeine, theobromine, and theophylline in these Standard Reference Materials (SRMs). The methods used at NIST to determine the concentration levels of caffeine, theobromine, and theophylline in SRM 3243 Ephedra-Containing Solid Oral Dosage Form and SRM 3244 Ephedra-Containing Protein Powder used reversed-phase liquid chromatography with absorbance detection and tandem mass spectrometry. These reference materials are part of the first suite in a series of NIST SRMs that provide concentration values for multiple components in dietary supplements. These SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and similar materials.     

Field-amplified on-line sample stacking for determination of carnosine-related peptides by capillary electrophoresis

An on-line sample stacking method, namely field-amplified sample injection, has been developed for the separation and determination of carnosine, anserine, and homocarnosine by capillary electrophoresis. Using electrokinetic injection, about 130- to 160-fold improvement of sensitivity was achieved without loss of separation efficiency when compared to conventional sample injection. For conventional injection, the samples were dissolved in running buffer and then hydrodynamically injected for 10 s (3.45 kPa). Various parameters affecting separation and sample stacking were optimized. Under optimum conditions, linear responses were obtained over two orders of magnitude and the detection limits (defined as S/N = 3) of carnosine, anserine, and homocarnosine were 1.5 x 10(-8) to 1.6 x 10(-8) mol/L.     

Ultrasound-assisted emulsification microextraction for the determination of ephedrines in human urine by capillary electrophoresis with direct injection. Comparison with dispersive liquid-liquid microextraction

Ultrasound-assisted emulsification microextraction and dispersive liquid-liquid microextraction were compared for extraction of ephedrine, norephedrine, and pseudoephedrine from human urine samples prior to their determination by capillary electrophoresis. Formation of a microemulsion of the organic extract with an aqueous solution (at pH 3.2) containing 10% methanol facilitated the direct injection of the final extract into the capillary. Influential parameters affecting extraction efficiency were systematically studied and optimized. In order to enhance the sensitivity further, field-amplified sample injection was applied. Under optimum extraction and stacking conditions, enrichment factors of up to 140 and 1750 as compared to conventional capillary zone electrophoresis were obtained resulting in limits of detection of 12-33 μg/L and 1.0-2.8 μg/L with dispersive liquid-liquid microextraction and ultrasound-assisted emulsification microextraction when combined with field-amplified sample injection. Calibration graphs showed good linearity for urine samples by both methods with coefficients of determination higher than 0.9973 and percent relative standard deviations of the analyses in the range of 3.4-8.2% for (n = 5). The results showed that the use of ultrasound to assist microextraction provided higher extraction efficiencies than disperser solvents, regarding the hydrophilic nature of the investigated analytes.     

Enhanced analysis of purine and pyrimidine bases by the use of double-strand polyaniline coatings in micellar electrokinetic capillary chromatography

A double-strand polymeric complex, which suppresses electroosmotic flow relative to fused-silica, is described. The polymeric complex contains a strand polyaniline (PAN) with the second strand containing polyacrylic acid (PAA) and methacrylate (MA) groups. The complex is referred to as PAN:P(AAMA). This polymeric complex has pH-controlled electroactive and hydrophobic characteristics and can be easily coated onto fused-silica. Enhanced separations of theophylline, theobromine, caffeine and adenine, thymine, uracil and cytosine were obtained by the use of the coated capillary in the micellar electrokinetic capillary electrophoresis (MEKC) system. The purine and pyrimdine bases were separated on the coated capillary with a 20 mM, pH 7 phosphate buffer which contains 0.05 M sodium dodecyl sulfate (SDS) as an additive.