Sensitive high-performance anion-exchange chromatographic determination of paeoniflorin and albiflorin by pulsed amperometric detection after solid-phase extraction

We developed a method to simultaneously determine paeoniflorin and albiflorin levels using high-performance anion-exchange liquid chromatography with pulsed amperometric detection (HPAEC-PAD). The main principle of our method includes solid-phase extraction step using Amberlite XAD-2 sorbent to remove sugars and to selectively determine glycosides by PAD. Under these conditions, the linear dynamic range was 0.01–100 μg/mL, and the albiflorin and paeoniflorin detection limits (S/N = 3) were 5 and 10 pg, respectively. The intra- and inter-day precisions (RSDs) were <5.07%, and the average recoveries from Paeoniae Radix and Si-ni-san ranged from 97.12 to 101.15%. Our method showed high selectivity, high sensitivity, and good repeatability for analyzing albiflorin and paeoniflorin in oriental medicinal preparation.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of phenylalanine in blood by high-performance anion-exchange chromatography–pulsed amperometric detection to diagnose phenylketonuria

We have developed a high-performance anion-exchange chromatography with pulsed amperometric detection method for the detection of phenylalanine (Phe) and diagnosis of phenylketonuria (PKU). Sample pretreatment steps were simplified without derivatization. The analyte was separated within 5 min. The detection limit (S/N = 3) for Phe was 50 pg. Linear dynamic range was 1.23–14.43 mg/dL (r² = 0.9999) for a dried blood spot. The mean recoveries of Phe for intra- and inter-day assays were found to be 96.87–104.16%. This method clearly differentiated PKU-positive groups from normal groups, and proved to be a practical procedure for rapid screening and follow-up monitoring of PKU.     

Sensitive high-performance liquid chromatography method of non-polar ginsenosides by alkaline-enhanced pulsed amperometric detection

We determined the minute amount of non-polar ginsenosides in red ginseng with a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method. Non-polar ginsenosides efficiently extracted by ethyl acetate were well separated in 40 min using a water–acetonitrile gradient eluent and detected by PAD under NaOH alkaline conditions. The ginsenoside detection limits (S/N = 3) were 0.03–0.10 ng. The coefficients of linear regression were 0.9972–0.9990. Intra- and inter-day precision (RSDs) was less than 8.34% and average recovery was 98.06–102.73%. The total amount of non-polar ginsenosides in hairy root of red ginseng was slightly higher than in the main root.     

Simultaneous determination of ascorbic acid,dopamine, uric acid and tryptophan on gold nanoparticles/overoxidized-polyimidazole composite modified glassy carbon electrode

A novel electrode was developed through electrodepositing gold nanoparticles (GNPs) on overoxidized-polyimidazole (PImox) film modified glassy carbon electrode (GCE). The combination of GNPs and the PImox film endowed the GNPs/PImox/GCE with good biological compatibility, high selectivity and sensitivity and excellent electrochemical catalytic activities towards ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp). In the fourfold co-existence system, the peak separations between AA–DA, DA–UA and UA–Trp were large up to 186, 165 and 285 mV, respectively. The calibration curves for AA, DA and UA were obtained in the range of 210.0–1010.0 μM, 5.0–268.0 μM and 6.0–486.0 μM with detection limits (S/N = 3) of 2.0 μM, 0.08 μM and 0.5 μM, respectively. Two linear calibrations for Trp were obtained over ranges of 3.0–34.0 μM and 84.0–464.0 μM with detection limit (S/N = 3) of 0.7 μM. In addition, the modified electrode was applied to detect AA, DA, UA and Trp in samples using standard addition method with satisfactory results.     

Rapid determination of Paeoniae Radix using near infrared spectroscopy

A rapid and nondestructive near infrared spectroscopy (NIRS) was used to differentiate different geographical Paeoniae Radix and quantitatively predict the content of main active components. Paeoniflorin, albiflorin and benzoylalbiflorin were analyzed simultaneously with an Agilent Zorbax SB-C₁₈ column by gradient elution under high-performance liquid chromatography-UV detection (HPLC-UV). Multiplicative scatter correction (MSC), first derivative and Savitsky-Golay were utilized together to correct the scattering effect and eliminate the baseline shift in all near infrared diffuse reflectance spectra in order to give a better correlation with the results obtained by HPLC-UV. Multiplicative regression methods were discussed. The spectra calibration equations produced highest correlation coefficient values (R²) and lowest root mean square error of prediction (RMSEP) were used for the determination of paeoniflorin, albiflorin and benzoylalbiflorin. The RMSEP of paeoniflorin, albiflorin and benzoylabiflorin were 0.866 mg/g, 0.369 mg/g and 0.084 mg/g, respectively, and the R² of cross validation were 0.986, 0.939 and 0.971, respectively. Furthermore with the use of principle component analysis (PCA), Paeoniae Radix was clustered according to different cultivation area. The results indicated that the NIRS method could be used for the quality control of Chinese herbal medicine.     

Determination of patulin in fruit juice and dried fruit samples by in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

A simple and sensitive method for the determination of patulin in fruit juice and dried fruit samples was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). Patulin was separated within 5 min by high-performance liquid chromatography using a Synergi MAX-RP 80A column and water/acetonitrile (80/20, v/v) as the mobile phase. Electrospray ionization conditions in the negative ion mode were optimized for MS detection of patulin. The pseudo-molecular ion [M−H]⁻ was used to detect patulin in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample using a Carboxen 1006 PLOT capillary column as an extraction device. The extracted patulin was readily desorbed from the capillary by passage of the mobile phase, and no carry-over was observed. Using the in-tube SPME LC–MS with SIM method, good linearity of the calibration curve (r = 0.9996) was obtained in the concentration range of 0.5–20 ng/mL using ¹³C₃-patulin as an internal standard, and the detection limit (S/N = 3) of patulin was 23.5 pg/mL. The in-tube SPME method showed >83-fold higher sensitivity than the direct injection method (10 μL injection volume). The within-day and between-day precision (relative standard deviations) were below 0.8% and 5.0% (n = 6), respectively. This method was applied successfully for the analysis of fruit juice and dried fruit samples without interference peaks. The recoveries of patulin spiked into apple juice were >92%, and the relative standard deviations were <4.5%. Patulin was detected at ng/mL levels in various commercial apple juice samples.     

Development and validation of a rapid assay based on liquid chromatography–tandem mass spectromtetry for determining macrolide antibiotic residues in eggs

A simple and rapid method able to determine residues of erythromycin A, tylosin and tilmicosin in whole eggs is presented here. The analytical protocol involves a one-step extraction followed by liquid chromatography (LC)–tandem mass spectrometry. Analytes were extracted from 1 g of egg spiked with an internal standard (josamycin) with acetonitrile. In terms of accuracy, matrix effect and ion signal stability, no extract cleanup was found to be necessary. After partial solvent removal, the final extract was injected into the LC column. Extraction was effective, since absolute recovery of the analyte in egg at their maximum residue limit (MRL) level was 85–102%. Estimated limits of quantification (S/N = 10) were 0.2–0.5 ng/g. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα) and detection capability (CCβ). The within-laboratory reproducibility, expressed as RSD (n = 18 at the MRL levels), was not higher than 13%. After validation, a short study on EA depletion in eggs was conducted after administration of this drug to laying hens.     

Simple assay for monitoring seven quinolone antibacterials in eggs: Extraction with hot water and liquid chromatography coupled to tandem mass spectrometry: Laboratory validation in line with the European Union Commission Decision 657/2002/EC

A simple and rapid method able to determine residues of seven quinolone antibacterials in whole eggs is presented here. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography–tandem mass spectrometry. After depositing 1.5 g of an egg sample containing the analytes and the analyte surrogate (norfloxacin) on sand (crystobalite), this material was packed into an extraction cell. Quinolones were extracted by flowing 6 mL of water acidified with 50 mmol/L formic acid through the cell heated at 100 °C. After pH adjustment and filtration of the extract, 100 μL of it was injected into the LC column. MS data acquisition was performed in the multiple reaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Hot water appeared an efficient extracting medium, since absolute recoveries of the analyte in egg at the level of 20 ng/g were 89–103%. Estimated limits of quantification (S/N = 10) were 0.2–0.6 ng/g. Based on the EU Commission Decision 2002/657/EC, the method was validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα and detection capability (CCβ). Depending on the particular analyte, CCαs ranged between 0.41 and 2.6 ng/g, while CCβs were 0.64–3.7 ng/g. The method was linear in the 3–30 ng/g range, with typical R² values higher than 0.97. The within-laboratory reproducibility (n = 21) at 6 ng/g level was in the 9.0–12% range. After validation, a depletion study of enrofloxacin and one of its metabolites, i.e. ciprofloxacin, in eggs was conducted.     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

Evaluation of the solid-phase microextraction fiber coated with single walled carbon nanotubes for the determination of benzene,toluene, ethylbenzene,xylenes in aqueous samples

A solid-phase microextraction (SPME) fiber coated with single walled carbon nanotubes (SWCNTs) was prepared by electrophoretic deposition and treated at 500 °C in H₂ stream. In order to evaluate the characteristics of the obtained fiber, it was applied in the headspace solid-phase microextraction (HS-SPME) of benzene, toluene, ethylbenzene and xylenes (BTEX) from water sample and quantification by gas chromatography with flame ionization detection (GC-FID). The results indicated that the thermal treatment with H₂ enhanced the extraction of the SWCNTs fiber for BTEX significantly. Thermal stability and durability of the fiber were also investigated, showing excellent stability up to 350 °C and life time over 120 times. In the comparison with the commercial CAR–PDMS fiber, the SWCNTs fiber showed similar and higher extraction efficiencies for BTEX. Under the optimized conditions, the linearity, LODs (S/N = 3) and LOQs (S/N = 10) of the method based on the SWCNTs fiber were 0.5–50.0, 0.005–0.026 and 0.017–0.088 μg/L, respectively. Repeatability for one fiber (n = 3) was in the range of 1.5–5.6% and fiber-to-fiber reproducibility (n = 3) was in the range of 4.2–8.3%. The proposed method was successfully applied in the analysis of BTEX compounds in seawater, tap water and wastewater from a paint plant.     

Simultaneous Determination of Paeoniflorin, Albiflorin and Benzoylpaeoniflorin in Radix Paeoniae Alba by TLC

Paeoniflorin, albiflorin and benzoylpaeoniflorin are three representative monoterpene glycosides in Radix Paeoniae Alba, a well-known traditional Chinese medicine with a great important biological activity. In the present paper, the three marker compounds were simultaneously quantified by TLC densitometric methods using high performance thin layer chromatography. The established method was validated in terms of LOD/LOQ, linearity, recovery and repeatability. The method was found to be precise with RSDs for intra-day in the range of 0.78–1.05, 0.67–0.98, 0.93–1.42% and for inter-day in the range of 0.85–1.23, 0.98–1.29, 1.28–1.94% for different concentrations of albiflorin, paeoniflorin and benzoylpaeoniflorin. Instrumental precision was 0.36, 0.41 and 0.45 (% RSD) for albiflorin, paeoniflorin and benzoylpaeoniflorin. Recoveries of abliflorinl, paeoniflorin and benzoylpaeoniflor were 98, 101.95 and 96.25%, respectively. The proposed method is simple, precise, specific, sensitive, and accurate and can be used for routine quality control of the crude drug.     

Solid phase microextraction using new sol–gel hybrid polydimethylsiloxane-2-hydroxymethyl-18-crown-6-coated fiber for determination of organophosphorous pesticides

A new sol–gel hybrid coating, polydimethylsiloxane–2-hydroxymethyl-18-crown-6 (PDMS–2OHMe18C6) was prepared in-house for use in solid phase microextraction (SPME). The three compositions produced were assessed for its extraction efficiency towards three selected organophosphorus pesticides (OPPs) based on peak area extracted obtained from gas chromatography with electron capture detection. All three compositions showed superior extraction efficiencies compared to commercial 100 μm PDMS fiber. The composition showing best extraction performance was used to obtain optimized SPME conditions: 75 °C extraction temperature, 10 min extraction time, 120 rpm stirring rate, desorption time 5 min, desorption temperature 250 °C and 1.5% (w/v) of NaCl salt addition. The method detection limits (S/N = 3) of the OPPs with the new sol–gel hybrid material ranged from 4.5 to 4.8 ng g⁻¹, which is well below the maximum residue limit set by Codex Alimentarius Commission and European Commission. Percentage recovery of OPPs from strawberry, green apple and grape samples with the new hybrid sol–gel SPME material ranged from 65 to 125% with good precision of the method (%RSD) ranging from 0.3 to 7.4%.     

A new capillary electrophoresis buffer for determining organic and inorganic anions in electroplating bath with surfactant additives

Monitoring of trace impurities in electroplating bath is needed to meet EU requirements for WEEE and RoHS and for quality control of electrodeposits. Methods using IC and 100% aqueous CE buffer were found producing non-repeatable results attributed to interference of surfactants and major methanesulphonate anion. A new CE buffer containing 1.5 mM tetraethylenepentaamine, 3 mM 1,3,5-benzenetricarboxylic acid and 15 mM Tris in 20% (v/v) methanol at pH = 8.4 was shown to enhance the separation window, reduce interaction between buffer and bath constituents, and give satisfactory repeatability with baseline separation for 14 organic and inorganic anions within 14 min, good repeatability for migration time (0.32–0.57% RSD), satisfactory peak area and peak height (2.9–4.5 and 3–4.7% respectively), low detection limit (S/N = 2, 20–150 ppb), and wide working ranges (0.1–100 ppm). The CE buffer with 20% (v/v) methanol has demonstrated its capability for identifying anion impurities causing problem in aged tin bath and the use of only 10-fold dilution to produce reliable results for quality assessment in plating bath containing high surfactant additives.     

Determination of aflatoxins in food samples by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

A simple and sensitive automated method for determination of aflatoxins (B1, B2, G1, and G2) in nuts, cereals, dried fruits, and spices was developed consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). Aflatoxins were separated within 8 min by high-performance liquid chromatography using a Zorbax Eclipse XDB-C8 column with methanol/acetonitrile (60/40, v/v): 5 mM ammonium formate (45:55) as the mobile phase. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of aflatoxins. The pseudo-molecular ions [M+H]⁺ were used to detect aflatoxins in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted aflatoxins were readily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC–MS with SIM method, good linearity of the calibration curve (r > 0.9994) was obtained in the concentration range of 0.05–2.0 ng/mL using aflatoxin M1 as an internal standard, and the detection limits (S/N = 3) of aflatoxins were 2.1–2.8 pg/mL. The in-tube SPME method showed >23-fold higher sensitivity than the direct injection method (10 μL injection volume). The within-day and between-day precision (relative standard deviations) at the concentration of 1 ng/mL aflatoxin mixture were below 3.3% and 7.7% (n = 5), respectively. This method was applied successfully to analysis of food samples without interference peaks. The recoveries of aflatoxins spiked into nuts and cereals were >80%, and the relative standard deviations were <11.2%. Aflatoxins were detected at <10 ng/g in several commercial food samples.     

Microwave-assisted on-line derivatization for sensitive flow injection fluorometric determination of formaldehyde in some foods

A rapid and sensitive flow injection fluorometry has been developed for the determination of formaldehyde based on the microwave on-line accelerating its Hantzsch reaction with cyclohexane-1,3-dione. Under the optimized conditions, the fluorescent intensity is proportional to formaldehyde content in the range from 0.05 ng/mL to 2.000 μg/mL. The detection limit (S/N = 3) is 0.02 ng/mL and the analytical frequency is 28 injections per hour. The relative standard deviations are 2.2% and 3.1% for eleven injections of 0.100 and 0.001 μg/mL of formaldehyde, respectively. With the assistance of microwave irradiation, a best sensitive fluorometry was established for the determination of formaldehyde at a high analytical frequency. This method was successfully applied to food analysis without requiring any sample pretreatment, and the determination results were correlated well with those obtained by the standard method with a sample pretreatment of steam distillation.     

Capillary electrophoresis-mass spectrometry using an in-line sol-gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid

In this study, a CE-MS method using a monolithic sol-gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R² > 0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N = 3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74-91%, demonstrating the promising potential of the methodology for the analysis of biological samples.     

Sandwich-type electrochemiluminescence immunosensor based on N-(aminobutyl)-N-ethylisoluminol labeling and gold nanoparticle amplification

A novel electrochemiluminescence (ECL) sandwich-type immunosensor for human immunoglobulin G (hIgG) on a gold nanoparticle modified electrode was developed by using N-(aminobutyl)-N-ethylisoluminol (ABEI) labeling. The primary antibody, goat-anti-human IgG was first immobilized on a gold nanoparticle modified electrode, then the antigen (human IgG) and the ABEI-labeled second antibody was conjugated successively to form a sandwich-type immunocomplex. ECL was carried out with a double-step potential in carbonate buffer solution (CBS) containing 1.5 mM H₂O₂. The ECL intensity increased linearly with the concentration of hIgG over the range 5.0-100 ng/mL. The limit of detection was 1.68 ng/mL (S/N = 3). The relative standard deviation was 3.79% at 60 ng/mL (n = 9). The present immunosensor is simple and sensitive. It has been successfully applied to the detection of hIgG in human serums.     

Determination of dye precursors in hair coloring products by liquid chromatography with electrochemical detection

The simultaneous determination of seven aminophenols, resorcinol and p-phenylenediamine in hair coloring products was performed by liquid chromatography (HPLC) with amperometric detection (ED). The aminophenols were separated on a ODS C18 reversed-phase column by isocratic elution with a mobile phase based on 0.1 M acetate buffer pH 4.5-methanol (90:10%, v/v) at a flow rate 0.8 mL min⁻¹. The limit of detection (S/N = 3) for the aminophenols was in the 15-40 pg (injected mass) range at an applied potential of 0.950 V versus Ag/AgCl. Peak heights for the aminophenols and the two others compounds were found to be linearly related to the amount injected, from 0.3 to 300 ng (r > 0.994-0.999).The relative standard deviation (R.S.D., n = 10) for 1 ng injected was comprised in the range from 2.5 to 6.2%, depending on the aminophenol tested. The present method minimizes troublesome and time-consuming pretreatment procedures and it was applied to the determination of aminophenols, resorcinol and phenylenediamine in hair coloring formulations.     

In situ synthesis of ceria nanoparticles in the ordered mesoporous carbon as a novel electrochemical sensor for the determination of hydrazine

A novel ceria (CeO₂)–ordered mesoporous carbon (OMC) modified electrode for the sensitive amperometric determination of hydrazine was reported. CeO₂–OMC composites were synthesized via a hydrothermal method at a relatively low temperature (180 °C) and characterized by scanning electron microscopy (SEM), transmission electron microcopy (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). The CeO₂–OMC modified glassy carbon electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) and indicated good electrocatalytic effect to the oxidation of hydrazine. Under the optimized conditions, the present sensor could be used to measure hydrazine in wide linear range from 40 nM to 192 μM (R² = 0.999) with a low detection limit of 12 nM (S/N = 3). Additionally, the sensor has been successfully applied to detect hydrazine in real water samples and the recoveries were between 98.2% and 105.6%. Eventually, the sensor exhibited an excellent stability and reproducibility as a promising method for determination of hydrazine.