Separation of enantiomers by packed capillary electrochromatography on a cellulose-based stationary phase

Separation of enantiomers was performed by applying packed capillary electrochromatography (CEC). Fused-silica capillaries of different lengths with an inner diameter of 100 microm were packed with a cellulose derivative immobilized onto macroporous silica gel. Parameters such as content of modifier in the mobile phase, concentration and pH of the buffer were varied for a set of test capillaries to determine their influence on enantioselectivity. In packed CEC the highest influence on resolution of the test racemates was found by changing the acetonitrile content, while variation of the buffer concentration mostly affects the electroosmotic velocity. The performance of packed CEC and nano-LC was also compared. Packed CEC showed much better column efficiency and enantioselectivity under similar flow/electroosmotic velocity.     

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700 ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine.     

In-tube solid-phase microextraction based on hybrid silica monolith coupled to liquid chromatography-mass spectrometry for automated analysis of ten antidepressants in human urine and plasma

A rapid, sensitive and automated in-tube solid-phase microextraction-liquid chromatography-mass spectrometry (in-tube SPME/LC-MS) method was developed for the analysis of ten antidepressants in urine and plasma. A hybrid organic-inorganic silica monolith with cyanoethyl functional groups was prepared and used as a sorbent for in-tube SPME. Integration of the sample extraction, LC separation and MS detection into a single system permitted direct injection of the diluted urine or plasma after filtration. Under the optimized conditions, good extraction efficiencies for the targets were obtained with no matrix interference in the subsequent LC-MS. Automation of the sampling, extraction and separation procedures was realized under the control of a program in this study. The total process time was 30 min and only 30 μL of urine or plasma was required in one analysis cycle. Good linearities were obtained for ten antidepressants with the correlation coefficients (R) above 0.9933. The limits of detection (S/N=3) for ten antidepressants were found to be 0.06-2.84 ng/mL in urine and 0.07-2.95 ng/mL in plasma. The recoveries of antidepressants spiked in urine and plasma were from 75.2% to 113.0%, with relative standard deviations less than 16.5%. The developed method was successfully used to analyze urine sample from ageing patients undergoing therapy with antidepressants.     

Immunoaffinity in-tube solid phase microextraction coupled with liquid chromatography-mass spectrometry for analysis of fluoxetine in serum samples

The inherent selectivity of the antibody was combined with in-tube solid-phase microextraction by immobilization of the antibody into the fused silica capillary. A sensitive, selective, and reproducible immunoaffinity in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry (in-tube SPME/LC-MS) method was developed, and validated for fluoxetine analysis in human serum. Important factors in the optimization of in-tube SPME variables, as well as the evaluation of the immunoaffinity capillary capacity are discussed. The in-tube SPME/LC-MS method presented a limit of quantitation of 5.00 ng/mL, and precision intra-assays with RSDs lower than 5%. The response of the in-tube SPME/LC-MS method for fluoxetine was linear over a dynamic range from 5.00 to 50.00 ng/mL, with correlation coefficients better than 0.998. Based on analytical validation it was demonstrated that in-tube SPME/LC-MS method offers high sensitivity, selectivity, and enough reproducibility to permit the quantification of fluoxetine in human serum at therapeutic levels. Thus, the proposed SPME/LC method can be useful tool to determine fluoxetine serum concentrations in patients receiving therapeutic dosages.     

Determination of stimulants in human urine and hair samples by polypyrrole coated capillary in-tube solid phase microextraction coupled with liquid chromatography-electrospray mass spectrometry

A simple and sensitive method for the determination of amphetamine, methamphetamine and their methylenedioxy derivatives in urine and hair samples was developed by coupling automated in-tube solid phase microextraction (SPME) to high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ES-MS). To achieve optimum performance, the conditions for both the in-tube SPME and the ES-MS detection were investigated. ES-MS detection conditions were studied by flow injection analysis (FIA) with direct liquid injection. In-tube SPME conditions were optimized by selecting the appropriate extraction parameters, including capillary stationary phases and sample pH. For the compounds studied, a custom-made polypyrrole (PPY) coated capillary showed superior extraction efficiency as compared to commercial capillaries. Therefore, the PPY coated capillary was selected for in-tube SPME in this study. The calibration curves of stimulants were linear in the range from 0.1 to 100 ng ml(-1) with detection limits (S/N=3) of 8-56 ng l(-1). This method was successfully applied to the analysis of the stimulants in spiked human urine and hair samples.     

Analysis of polar pesticides in water and wine samples by automated in-tube solid-phase microextraction coupled with high-performance liquid chromatography-mass spectrometry

A simple and sensitive method for the determination of polar pesticides in water and wine samples was developed by coupling automated in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). To achieve optimum performance, the conditions for both the in-tube SPME and the ESI-MS detection were investigated. In-tube SPME conditions were optimized by selecting the appropriate extraction parameters, especially the stationary phases used for SPME. For the compounds studied, a custom-made polypyrrole (PPY)-coated capillary showed superior extraction efficiency as compared to several commercial capillaries tested, and therefore, it was selected for in-tube SPME. The influence of the ethanol content on the performance of in-tube SPME was also investigated. It was found that the amount of pesticides extracted decreased with the increase of ethanol content in the solutions. The ESI-MS detection conditions were optimized as follows: nebulizer gas, N2 (30 p.s.i.; 1 p.s.i.=6894.76 Pa); drying gas, N2 (10 l/min, 350 degrees C); capillary voltage, 4500 V; ionization mode, positive; mass scan range, 50-350 amu; fragmentor voltage, variable depending on the ions selected. Due to the high extraction efficiency of the PPY coating and the high sensitive mass detection, the detection limits (S/N = 3) of this method for the compounds studied are in the range of 0.01 to 1.2 ng/ml, which are more than one order of magnitude lower than those of the previous in-tube SPME-HPLC-UV method. A linear relationship was obtained for each analyte in the concentration range of 0.5 to 200 ng/ml with MS detection. This method was applied to the analysis of phenylurea and carbamate pesticides in spiked water and wine samples.     

In-tube solid-phase microextraction with poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary for direct high-performance liquid chromatographic determination of ketamine in urine samples

Ketamine was used for anaesthesia originally but has emerged as an abused drug in recent years. The prevalence of ketamine abuse demands a direct and rapid determination method. It is known that in-tube solid phase microextraction (in-tube SPME) can perform extraction with a capillary linked directly to a HPLC system, providing an automated and accurate extraction procedure. In this paper, an in-tube SPME coupled to HPLC method was developed for the determination of ketamine in urine samples with a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column as the extraction phase, which is expected to provide higher extraction efficiency than open tubular capillaries. After optimizing the extraction conditions, ketamine was extracted directly from urine samples in a wide dynamic linear range of 50-10,000 ng mL(-1), with the detection limit obtained as 6.4 ng mL(-1). The intra-day and inter-day precision for the method was 1.6% and 1.7%, respectively. The urine samples from suspect addicts have been successfully analyzed within 20 min. The re-usability of the monolithic column was also confirmed as no decrease of the extraction efficiency was shown after urine extraction.     

Automated in-tube solid phase microextraction coupled with HPLC-ES-MS for the determination of catechins and caffeine in tea

A polypyrrole (PPY) coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for catechins and caffeine. Compared with commercial capillaries that were currently used for in-tube solid phase microextraction (SPME), the PPY coated capillary showed better extraction efficiency for all of the compounds studied. Electrospray mass spectrometric (ES-MS) detection conditions were also investigated. After optimization of the extraction and detection conditions, a method for the sensitive and selective determination of catechins and caffeine was developed by coupling the PPY coated capillary in-tube SPME with HPLC-ES-MS. Catechins could be determined in both positive and negative ion detection modes. The detection limit (S/N = 3) for each of the studied catechins was < 0.5 ng mL-1. Caffeine could only be determined under positive ES-MS detection conditions and its detection limit was 0.01 ng mL-1. Caffeine and the five catechins in several tea samples were determined using the developed method. Small amounts of catechins were also detected in grape juice and wine samples.     

Automated analysis of lidocaine and its metabolite in plasma by in-tube solid-phase microextraction coupled with LC-UV for pharmacokinetic study

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for determination of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in human plasma has been developed, validated, and further applied to pharmacokinetic study in pregnant women with gestational diabetes mellitus (GDM) subjected to epidural anesthesia. Important factors in the optimization of in-tube SPME performance are discussed, including the draw/eject sample volume, draw/eject cycle number, draw/eject flow rate, sample pH, and influence of plasma proteins. The limits of quantification of the in-tube SPME/LC method were 50 ng/mL for both metabolite and lidocaine. The interday and intraday precision had coefficients of variation lower than 8%, and accuracy ranged from 95 to 117%. The response of the in-tube SPME/LC method for analytes was linear over a dynamic range from 50 to 5000 ng/mL, with correlation coefficients higher than 0.9976. The developed in-tube SPME/LC method was successfully used to analyze lidocaine and its metabolite in plasma samples from pregnant women with GDM subjected to epidural anesthesia for pharmacokinetic study.     

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.     

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.     

Monolithic silica column for in-tube solid-phase microextraction coupled to high-performance liquid chromatography

In-tube solid-phase microextraction (SPME) has successfully been coupled to capillary LC, and further an automated in-tube SPME system has been developed using a commercially available HPLC auto-sampler. However, an open tubular capillary column with a thick film of polymer (stationary phase) is unfavorable because the ratio of the surface area of coating layer contacted with sample solution to the volume of the capillary column is insufficient for mass transfer. A highly efficient SPME column is. therefore, required. We introduced a C18-bonded monolithic capillary column that was used for in-tube SPME. The column consisted of continuous porous silica having a double-pore structure. Both the through-pore and the meso-pore were optimized for in-tube SPME, and the optimized capillary column was connected to an HPLC injection valve for characterization. The results demonstrated that the pre-concentration efficiency is excellent compared with the conventional in-tube SPME. The novel method for both introduction and concentration of the samples was effective. satisfactory and suitable for use in the SPME medium.     

Determination of camptothecin and 10-hydroxycamptothecin in human plasma using polymer monolithic in-tube solid phase microextraction combined with high-performance liquid chromatography

A biocompatible in-tube solid-phase microextraction (SPME) device was used for the direct and on-line extraction of camptothecin and 10-hydroxycamptothecin in human plasma. Biocompatibility was achieved through the use of a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column for extraction. Coupled to high performance liquid chromatography (HPLC) with UV detection, this on-line in-tube SPME method was successfully applied to the simultaneous determination of camptothecin and 10-hydroxycamptothecin in human plasma. The calculated detection limits for camptothecin and 10-hydroxycamptothecin were found to be 2.62 and 1.79 ng/mL, respectively. The method was linear over the range of 10–1000 ng/mL. Excellent method reproducibility was achieved, yielding RSDs of 2.49 and 1.59%, respectively. The detection limit (S/N=3) of camptothecin was found to reach 0.1 ng/mL using fluorescence detection. The proposed method was shown to cope robustly with the extraction and analysis of camptothecin and 10-hydroxycamptothecin in plasma samples.     

Determination of enantiomers of atenolol and propranolol in pharmaceutical formulations by HPLC

A validated HPLC-UV method was developed for the determination of R(-), S(+)-atenolol and R(-), S(+)-propranolol in pharmaceutical formulations. The proposed method required no elaborate sample preparation and was found to be selective, linear, and repeatable within the established ranges. Atenolol and propranolol isomers were separated using a Chirex 3022 (S) column with the mobile phases hexane-dichloromethane-methanol-trifluoroacetic acid (35 + 35 + 5 + 0.25, v/v/v/v) and hexane-dichloromethane-ethanol-trifluoroacetic acid (55 + 40 + 5 + 0.25, v/v/v/v), respectively. The LOD values of R(-) and S(+)-atenolol were 12.3 and 9.86 microg/mL, respectively, and 0.61 and 0.89 microg/mL, respectively, for R(-) and S(+)-propranolol. Retention times of R(-)-propranolol and S(+)-propranolol were 12.4 and 14.3 min, respectively, and 29.06 and 32.71 min, respectively, for (R)-atenolol and (S)-atenolol. The proposed method was applied to the determination of enantiomers in pharmaceutical formulations, and no interference from any excipients was found.     

Determination of fluoroquinolones in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

We developed a sensitive and useful method for the determination of five fluoroquinolones (FQs), enoxacin, ofloxacin, ciprofloxacin, norfloxacin, and lomefloxacin in environmental waters, using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These compounds were analysed within 7 min by high-performance liquid chromatography (HPLC) using a CAPCELL PAK C8 column and aqueous ammonium formate (pH 3.0, 5 mM)/acetonitrile (85/15, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection. In order to optimize the extraction of FQs, several in-tube SPME parameters were examined. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample at a flow-rate of 150 μL/min, using a Carboxen 1010 PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r ≥ 0.997) was obtained in the concentration range from 0.1 to 10 ng/mL for all compounds examined. The limits of detection (S/N = 3) of the five FQs ranged from 7 to 29 pg/mL. The in-tube SPME method showed 60-94-fold higher sensitivity than the direct injection method (5 μL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface waters and wastewaters were collected from the area around Asahi River, and ofloxacin was detected in wastewater samples of a sewage treatment plant and other two hospitals at 17.5-186.2 pg/mL. The recoveries of FQs spiked into river water were above 81% for a 0.1 or 0.2 ng/mL spiking concentration, and the relative standard deviations were below 1.9-8.6%.     

管内硼亲和固相微萃取-高效液相色谱全自动在线联用检测茶饮料中的顺式二羟基化合物

自动化联用分析技术对于降低人力强度、提高效率和保证数据重现性等具有重要意义。硼亲和固相微萃取（BA-SPME）是近十年出现的用于富集顺式二羟基化合物的独特工具,但BA-SPME与高效液相色谱（HPLC）的自动化在线联用还未见报道。该文报道了一种新颖的管内BA-SPME-HPLC全自动在线联用方法,用于分析茶饮料中的顺式二羟基化合物。该自动化在线联用方法利用自动进样器通过六通阀的切换实现流路连接。制备了管内BA-SPME毛细管,考察了涂层柱的柱容量,并对其形貌进行了表征,考察并优化了影响实际样品分离效果的因素。最后,利用该联用方法对3种不同品牌的茶饮料进行了分析,并对沏茶温度对茶水中顺式二羟基化合物含量的影响进行了评价。     

Quantification of propranolol enantiomers in small blood samples from rats by reversed-phase high-performance liquid chromatography after chiral derivatization

A high-performance liquid chromatographic (HPLC) technique is described for quantification of R(+)- and S(-)-propranolol from 100-microliters rat blood samples. The procedure involves chiral derivatization with tert.-butoxycarbonyl-L-leucine anhydride to form diastereomeric propranolol-L-leucine derivatives which are separated on a reversed-phase HPLC column. The method as previously reported has been modified for assaying serial blood microsamples obtained from the rat for pharmacokinetic studies. An internal standard, cyclopentyldesisopropylpropranolol, has been incorporated into the assay and several derivatization parameters have been altered. Standard curves for both enantiomers were linear over a 60-fold concentration range in 100-microliters samples of whole rat blood (12.5-750 ng/ml; r = 0.9992 for each enantiomer). Inter- and intra-assay variability was less than 12% for each enantiomer at 25 ng/ml. No enantiomeric interference or racemization was observed as a result of the derivatization. No analytical interference was noted from endogenous components in rat blood samples. Preliminary data from two male Sprague-Dawley rats given a 2.0 mg/kg intravenous dose of racemic propranolol revealed differential disposition of the two enantiomers. R(+)-Propranolol achieved higher initial concentration but was eliminated more rapidly than S(-)-propranolol. Terminal half-lives of R(+)- and S(-)-propranolol were 19.23 and 51.95 min, respectively, in one rat, and 14.50 and 52.07 min, respectively, in the other.     

Enantioselective drug monitoring of (R)- and (S)- propranolol in human plasma via derivatization with optically active (R,R)-O,O-diacetyl tartaric acid anhydride

A sensitive high-performance liquid chromatographic method was developed for the stereoselective assay of (R)- and (S)-propranolol in human plasma. The method involves diethyl ether extraction of the drugs and a racemic internal standard, N-tert.-butylpropranolol, followed by derivatization of the compounds with the chiral reagent (R,R)-O,O-diacetyl tartaric acid anhydride. The resulting diastereomeric derivatives were separated isocratically on a reversed-phase column. Quantitation was achieved by the peak-height ratio method with reference to the internal standard. The assay was accurate and reproducible in the concentration range 1-100 ng of (R)- and (S)-propranolol per ml plasma, using fluorescence detection at lambda ex 290 nm and lambda em 335 nm. The applicability of this method was demonstrated for the determination of concentration-time profiles of propranolol enantiomers in the course of comparative pharmacokinetic studies.     

Hybrid organic-inorganic octyl monolithic column for in-tube solid-phase microextraction coupled to capillary high-performance liquid chromatography

An octyl-functionalized hybrid silica monolithic column was developed for in-tube solid-phase microextraction (SPME) to perform on-line preconcentration coupled to capillary high-performance liquid chromatography (microHPLC) analysis. A hybrid silica monolithic column functionalized with octyl groups was conveniently synthesized by a two-step acid/base-catalyzed hydrolysis/co-condensation of tetraethoxysilane (TEOS) and n-octyltriethoxysilane (C8-TEOS). The size of through-pores as well as the carbon content can be adjusted by changing the ratio of TEOS to C8-TEOS in the polymerization mixture. The extraction characteristics of the monolithic column prepared under optimized fabrication conditions were studied by using polycyclic aromatic hydrocarbons (PAHs) as the analytes. The sample volume that could be injected into the system was increased up to 1mL with simultaneous increase of column efficiency, when hybrid silica monolithic column was used as a precolumn. Good linear calibration curves (R>0.999) were obtained, and the limits of detection (signal-to-noise ratio, S/N=3) for the analytes were found to be between 2.4 and 8.1ng/mL with a UV absorbance detector, which are 299-456 times lower than those obtained without preconcentration. The column-to-column RSD values were 1.3-8.0% for recoveries of PAHs investigated.     

An efficient slurry packing procedure for the preparation of columns applicable in capillary electrochromatography and capillary electrochromatography‐electrospray‐mass spectrometry

A procedure is described for the slurry packing of 50‐μm ID fused silica capillaries with 3‐μm octadecyl silica (ODS) particles for capillary electrochromatography (CEC) and its hyphenation with electrospray ionisation mass spectrometry (ESI/MS). A homogeneous packed bed is obtained by using a slow packing process in an upward direction with a balanced density slurry solvent and MeOH as packing solvent. Special attention was paid to the in‐ and outlet frit preparation in order to avoid gas bubble formation which renders CEC‐ESI/MS problematic. Frits were made out of the packed bed itself, sintered in water, by using a perforated heating ribbon; they were not longer than 1 mm. In CEC‐UV, column efficiencies up to 300,000 plates per meter were obtained. Absence of gas bubbles was ascertained by the straightforward coupling to ESI/MS. A make‐up flow of 3 μL/min H₂O/MeOH containing 0.1% HCOOH was used in the sheath flow interface. Steroids and carbamates were analysed with a 0.1% triethylamine‐acetic acid buffer (pH 8.9) containing varying amounts of acetonitrile. In CE‐ESI/MS, efficiencies dropped by ca. 20% but spectral data were excellent.