Determination of benzo[a]pyrene tetrols by column-switching capillary liquid chromatography with fluorescence and micro-electrospray ionization mass spectrometric detection

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzo[a]pyrene tetrol isomers in biological matrices using on-line fluorescence and micro-electrospray ionization mass spectrometric detection. A well-established off-line crude solid phase extraction procedure was used in order to make the method compatible with several biological matrices. The solid phase extraction eluates were evaporated to dryness, redissolved in 1.0 ml methanol:water (10:90, v/v), loaded onto a 0.32 mm I.D. x 40 mm 5 microm Kromasil C(18) pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. x 150 mm 3.5 microm Kromasil C(18) analytical column. The samples were loaded with a flow rate of 50 microl min(-1) and the tetrols were separated at a flow rate of 4 microl min(-1) with an acetonitrile:10 mM ammonium acetate gradient from 10 to 90%. A sample loading flow rate up to 50 microl min(-1) was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzo[a]pyrene tetrols was obtained by monitoring their [M - H](-) molecular ions at m/z 319. The method was validated over the concentration range 0.1-50 ng ml(-1) benzo[a]pyrene tetrols in a cell culture medium with 100 microl injection volume, fluorescence detection and the first eluting tetrol isomer as model compound, resulting in a correlation coefficient of 0.993. The within-assay (n= 6) and between-assay (n= 6) precisions were determined to 2.6-8.6% and 3.8-9.6%, respectively, and the recoveries were determined to 97.9-102.4% within the investigated concentration range. The mass limit of detection (by fluorescence) was 3 pg for all the tetrol isomers, corresponding to a concentration limit of detection of 30 pg ml(-1) cell culture medium. The corresponding mass spectrometric mass limits of detection were 4-10 pg, corresponding to concentration limits of detection of 40-100 pg ml(-1) cell culture medium.     

Trace analysis of pesticide residues in water by high-speed narrow-bore capillary gas chromatography-mass spectrometry with programmable temperature vaporizer

A method for the rapid trace analysis of 17 residual pesticides in water by narrow-bore capillary (I.D. 100 microm) gas chromatography-mass spectrometry (GC-MS) using a programmable temperature vaporizer (PTV) was discussed. The method consisted of a large-volume injection (40 microl) by a PTV, high-speed analysis using a narrow-bore capillary column and MS detection. The PTV with solvent vent mode was very useful for large-volume injection into a narrow-bore capillary column because the injected solvent volume could be reduced to less than 2 microl. The analysis time was 8.5 min [less than 50% of the analysis time using conventional columns (I.D. 250 microm)]. A 10-ml volume of river water was extracted by dichloromethane (4 ml), and then the extract was condensed to 1 ml. This extract was analyzed. Mean recoveries for river water spiked at 100 pg/ml ranged from 83.4 to 96.7%. The limit of detections of the 17 pesticides ranged from 1 to 100 pg/ml.     

On-line SPE-Nano-LC-Nanospray-MS for rapid and sensitive determination of perfluorooctanoic acid and perfluorooctane sulfonate in river water

An instrumental set up including on-line solid-phase extraction, nano-liquid chromatography, and nanospray mass spectrometry is constructed to improve the sensitivity for quantitation of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in surface water. Sample volumes of 1000 microL are loaded onto a microbore 1.0-mm i.d. x 5 mm, 5 microm Kromasil C(18) enrichment column by a carrier solution consisting of 10mM ammonium acetate in acetonitrile-water (10:90, v/v) at a flow rate of 250 microL/min, providing on-line analyte enrichment and sample clean-up. Backflushed elution onto a 0.1-mm i.d. x 150 mm, 3.5 microm Kromasil C(18) analytical column is conducted using an acetonitrile-10mM ammonium acetate solvent gradient from 30% to 70% acetonitrile. Water samples are added with internal standard (perfluoroheptanoic acid) and filtrated prior to injection. The mass limits of detection of PFOA and PFOS are 0.5 and 1 pg, respectively, corresponding to concentration limits of detection of 500 pg/L and 1 ng/L, respectively. The total time spent on sample preparation, chromatography, and detection is approximately 12 min per sample. The method was employed for the determination of PFOS and PFOA in urban river water.     

Temperature optimization for improved determination of phosphatidylserine species by micro liquid chromatography with electrospray tandem mass spectrometric detection

A sensitive method for determination of disaturated phosphatidylserine species in the presence of their monounsaturated analogs has been developed, using micro liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The hydrophobic nature of the phosphatidylserine species required a combination of low-eluting sample solvents and sub-ambient temperatures in order to focus large sample volumes up to 20 microL. The samples were dissolved in 2-propanol:hexane:water (20:10:4, v/v/v) prior to 1:9 dilution with ammonium formate buffer:2-propanol:tetrahydrofuran (30:55:15, v/v/v) and final 1:4 dilution with ammonium formate buffer (10 mM):2-propanol: tetrahydrofuran (55:37.5:7.5, v/v/v). The analytical column was a 0.5 x 150 mm stainless steel column packed with 5 microm C30 particles, while the mobile phase contained ammonium formate buffer (10 mM): 2-propanol:tetrahydrofuran (30:55:15, v/v/v). A temperature program from 5 degrees C (hold for 3 minutes) to 75 degrees C at 8 K/min provided separation of the disaturated phosphatidylserine species from their monounsaturated analogs, making available a sensitive determination of the isobaric species. The mass limit of detection for dipalmitoyl phosphatidylserine was 100 pg, corresponding to a concentration limit of detection of 5 pg/microL when using an injection volume of 20 microL. This is an improvement by a factor of 20 as compared to previously reported numbers obtained with conventional LC columns. The within-assay precision of dipalmitoyl phosphatidylserine was 11.9% RSD (n = 3), while the retention time precision was 4.1% RSD (n = 6).     

Determination of low levels of an antioxidant in polyolefins by large-volume injection temperature-programmed packed capillary liquid chromatography

Sub-ambient column temperatures, promoting strong interactions between the analyte and the stationary phase material, were utilized to focus large volumes of the polyolefin antioxidant Irganox 1076 [benzenepropanoic acid, 3.5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester] on the column inlet, using pure acetonitrile as sample solvent and mobile phase. Injection volumes up to 100 microl were successfully employed on a 50 cm x 320 microm I.D. capillary column packed with 5 microm Kromasil 100 ODS particles. Irganox 1076 was eluted after completed injection by temperature programming, using a temperature program from 7 to 90 degrees C, in 3 degrees C min(-1). UV detection, using a low-dispersion "U"-shaped flowcell, was performed at 280 nm. The method was applied for the determination of Irganox 1076 that was extracted from low-density polyethylene (0.6 ppm, w/w). Both Soxhlet and microwave-aided solvent extractions were performed, using chloroform and acetonitrile as solvents, respectively. The microwave-aided extraction with acetonitrile was found to give approximately the same yield as the standard Soxhlet reference method. Consequently, small volumes of acetonitrile could be used both as extraction solvent, sample solvent and mobile phase, simplifying the analysis process. The mass limit of detection of the method was found to be 3.3 ng, corresponding to a concentration limit of detection of 33 ng ml(-1), utilizing an injection volume of 100 microl. The within and between day precision of retention times displayed relative standard deviations below 1.2%.     

Determination of polyethylene glycol in low-density polyethylene by large volume injection temperature gradient packed capillary liquid chromatography

Polyethylene glycol (PEG) 20000 in low-density polyethylene has been determined using column switching and inverse temperature programming in reversed-phase packed capillary liquid chromatography with evaporative light scattering detection. PEG 20000 was extracted into water from the polyethylene dissolved in toluene and PEG 35000 was added as an internal standard (I.S.). The samples in aliquots of 100 microl were reconcentrated on the enrichment column using a loading mobile phase of acetonitrile-water (3:97, v/v) at a flow-rate of 75 microl/min for 3 min, then back-flushed and separated on the analytical column with acetonitrile-THF-water (40:5:55, v/v) as mobile phase. The column temperature was reduced from 68 to 55 degrees C with a ramp of -1.5 degrees C/min, held constant for 3 min and then reduced further to 45 degrees C with a -1.5 degrees C/min ramp and kept constant for 1 min. The analysis runtime was 20 min. The recovery of PEG 20 000 was determined to 65.1% with 2.8% RSD and the mass limit of detection of PEG 20 000 was 1.25 microg. The within-assay and between day precision of the retention times of both PEG 20000 and PEG 35000 displayed RSD of less than 1.1% (n = 9), while the overall area ratio RSD of 100 microg/ml PEG 20000 over PEG 35000 was 1.3% (n = 9). The method was linear within the investigated concentration range 25-125 microg/ml (R2 = 0.9983). In addition, a mixture of PEG 4000, 8000, 10000, 20000 and 35000 was analysed on the system to demonstrate the possibility of analysing several PEGs in a sample with the use of temperature gradient elution.     

Determination of 1-(2-methoxyphenyl)piperazine derivatives of isocyanates at low concentrations by temperature-programmed miniaturized liquid chromatography

A temperature-programmed packed capillary LC method with large-volume injection on-column focusing has been developed for screening and determination of 1-(2-methoxyphenyl)piperazine derivatives of airborne toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, hexamethylenediisocyanate and methylenebisphenyl-4,4-diisocyanate, based on sampling methods described in MDHS 25/3. Injection volumes up to 100 microl were successfully loaded onto the 250x0.32 mm I.D. capillary column packed with 3 microm Hypersil ODS particles. The isocyanate derivatives were loaded at 10 degrees C and eluted by a three-step temperature program starting at 10 degrees C for 10 min, followed by a temperature ramp of 2.5 degrees C min(-1) to 45 degrees C and then 9.9 degrees C min(-1) to 90 degrees C. The mobile phase consisted of acetonitrile-acetate buffer (3% triethylamine, pH 4.5) (45:55, v/v). The isocyanate derivatives were dissolved in acetonitrile-acetate buffer (3% triethylamine, pH 4.5) (30:70, v/v) to achieve sufficient focusing. The concentration limit of detection of the individual derivatives utilizing an "U" shaped flow cell with a 8.0 mm light path and an injection volume of 100 microl was 44, 87, 43 and 210 pg ml(-1) for toluene-2,6-diisocyanate, hexamethylenediisocyanate, toluene-2,4-diisocyanate and methylenebisphenyl-4,4-diisocyanate, respectively. Within the investigated concentration range, 10-500 ng ml(-1), the linear calibration curves gave correlation coefficients ranging from 0.994 to 0.998. The repeatability of the method with regard to retention time and peak height ranged from 0.3 to 1.1% and 1.1 to 2.3% (n=9) relative standard deviation, respectively. The average recovery of the method, with regard to toluene-2,4-diisocyanate, was 97.7+/-1.6% (n=9).     

Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.     

Limitations of porous graphitic carbon as stationary phase material in the determination of catecholamines

A fast and sensitive capillary liquid chromatography (cLC) column-switching method with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection for the simultaneous determination of dopamine (D), epinephrine (E), norepinephrine (NE) and serotonin (SE) was pursued. A sample volume of 100 microl was loaded with a mobile phase containing 0.1% pentafluoropropionic acid (PFPA) as ion-pairing agent on a 25 mm x 0.32 mm (i.d.) 5 microm Hypercarb column. A water-acetonitrile (AcN) gradient with 0.1% acetic acid (AcOH) backflushed the compounds onto a 34 mm x 0.32 mm (i.d.) 5 microm Hypercarb analytical column. However, during a series of analyses, oxidation of the catecholamines (CAs) was observed. This was suspected to be due to the loading mobile phase composition and precluded the usefulness of this method even though the achievable detection limit was in the range of 0.75-3.0 ng/ml. The combination of the porous graphitic carbon (PGC) material and the fluorinated strong acids which were required to get enough retention for preconcentration of large volumes cannot be used for easily oxidized compounds as the CAs.     

Determination of perfluorooctane sulfonate and perfluorooctanoic acid in human plasma by large volume injection capillary column switching liquid chromatography coupled to electrospray ionization mass spectrometry

Rapid, selective, and sensitive methodology for the quantification of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in human plasma using packed capillary liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry has been developed. Plasma proteins were precipitated using acetonitrile and the resulting supernatant was diluted 1+1 with water containing 10 mM ammonium acetate (NH4Ac) prior to injection. Sample volumes of 250 microL were loaded onto a 30 mm x 0.32 mm ID 10 microm Kromasil C18 precolumn by a carrier solution consisting of 10 mM NH4Ac in ACN/H2O (5/95, v/v) at a flow rate of 100 microL/min, providing on-line analyte enrichment and sample clean-up. Backflushed elution onto a 100 mm x 0.32 mm ID 3.5 microm Kromasil C18 analytical column was conducted using an ACN/H2O solvent gradient containing 10 mM NH4Ac. In order to improve the robustness and performance of the method, perfluoroheptanoic acid (PFHA) was used as internal standard. Separation and detection of PFOA, PFHA, and PFOS were achieved within 10 minutes. Ionization was performed in the negative mode in the m/z range 250-550. The method was validated over the concentration range 1-200 ng/mL for PFOA and over the range 5-200 ng/mL untreated plasma for PFOS, yielding correlation coefficients of 0.997 (PFOA) and 0.996 (PFOS), respectively. The within-assay (n = 6) and between-assay (n = 6) precisions were in the range 2.1-9.2 and 5.6-12%, respectively. The concentration limits of detection (cLOD) of PFOA was 0.5 ng/mL while the cLOD of PFOS was estimated to be 0.2 ng/mL in untreated plasma.     

Determination of bradykinin and arg-bradykinin in rat muscle tissue by microdialysis and capillary column-switching liquid chromatography with mass spectrometric detection

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.     

Online trace enrichment to determine pyrethroids in river water by HPLC with column switching and photochemical induced fluorescence detection

The potential of online trace enrichment on a highly apolar short column in LC was evaluated for the determination of pyrethroids in river water. Twelve millilitres of water samples, modified with 8 mL ACN (ACN/water 40:60, v/v), were passed through 50 x 4.6 mm ID first separation column packed with 5 microm Hypersil Elite C18. Pesticides were preconcentrated in this column while the matrix background was eluted to waste. Separation of pesticides was performed on a 3.5 microm symmetric C18 column (250 x 4.6 mm ID) with an ACN step gradient as mobile phase and fluorescence detection was used after postcolumn derivatization by using UV light. The use of photochemically induced fluorescence for detection improved sensitivity and selectivity. Quantification limits ranged from 0.05 to 0.1 microg/L and pesticide recoveries at two concentration levels (0.1 and 0.5 microg/L) were between 93.1 and 118.6%, with RSD between 2.5 and 7.5% (n = 3) in river water samples. No matrix effect was detected.     

Sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry

The development of an on-column focusing gradient capillary LC method coupled to tandem mass spectrometry (quadrupole-linear ion trap) for the quantitative determination of the anticancer agent ZD1839 (Gefitinib, Iressa) in blood plasma is described. Plasma samples (0.2 ml) were extracted with methyl tert-butyl ether. The analytes of interest, ZD1839 and the internal standard [(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm x 1 mm, 5 microm particle size, capillary ODS Hypersil column using an aqueous ammonium acetate gradient at 40 microl/min. Mass spectrometric detection was performed by a Q-Trap tandem mass spectrometer with electrospray positive ionisation, and monitored in the multiple reaction monitoring transitions 447 >128 and 455 >136, respectively. The limit of quantification of ZD18395 was 0.1 ng/ml. The method proved to be robust, allowing quantification of ZD1839 with sufficient precision, accuracy and sensitivity.     

填充毛细管液相色谱-毛细管气相色谱在线联用分析八角茴香挥发油

首次将填充毛细管高效液相色谱-毛细管气相色谱在线联用技术(μ-HPLC-CGC)用于分离分析八角茴香果实的挥发油成分。液相色谱选用氰基分析柱(250 mm×0.32 mm i.d.),正己烷-乙腈-二氯甲烷(体积比为80∶8∶12)为流动相,对挥发油样品做族组分分离,得到的5个族组分被依次存放在多位储存接口内,然后不分流分别转入毛细管气相色谱仪做详细分析。气相色谱柱由10 m×0.53 mm i.d.保留间隔柱和30 m×0.53 mm i.d.×1.0 μm SE-54分析柱组成。采用了不分流柱内进样模     

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).     

Microchip-based capillary electrochromatography using packed beds

Integration of a packed column onto a microchip for performance of capillary electrochromatography (CEC) is described. The quartz device incorporated a cross-injector, and a double weir trapping design for formation of 1, 2 and 5 mm long CEC columns. Three fluorescent dyes were baseline-resolved with plate numbers of 330,(330,000 plates/m; height equivalent to a theoretical plate, H = 3.0 microm) for BODIPY 493/503, 360 (360,000 plates/m; H = 2.8 microm) for rhodamine 123 and 244 (244,000 plates/m; H = 4.1 microm) for acridine orange (AO) with 500 V applied on a 1 mm long column. The 2 mm column yielded approximately 1.8 times more theoretical plates than did the 1 mm column, when operated at the same flow rate. Van Deemter plots were obtained for the three column lengths, showing increased plate height for the 5 mm length. A 2 mm column gave peak height and area relative standard deviation (RSD) values of 2.5 and 3.3%, respectively, as averages for the three dyes (n = 15). The RSD for the dye retention times was 1% (n = 6) over one day, and 3% (n = 30) over five days. Indirect fluorescence detection of thiourea and of amino acids was possible using a neutral indicator dye (BODIPY 493/503), with a detection limit of 10 microM for amino acids.     

AQC柱前衍生化RP-HPLC法测定蒜氨酸及其有关物质的含量

采用6-氨基喹啉-N-(羟基琥珀酰亚胺基)氨基甲酸酯(6-aminoquinolyl -N- Hydroxysuccinimide Carbamate ,AQC)为柱前衍生化试剂,建立了AQC柱前衍生化RP-HPLC法测定蒜氨酸及其有关物质的含量。该衍生化方法反应瞬间完成,衍生化产物稳定。色谱条件为：Kromasil C18柱（250mm×4.6mm,5mm）,流动相A为0.1%乙酸铵(含0.03%乙酸),流动相B为水-乙腈（40∶60）,线性梯度洗脱,流速1.0ml/min,检测波长248nm。蒜氨酸在1.1719~1500μg /ml浓度范围内线性关系良好（r=0.9998）, 日内、日间精密度良好(RSD <1.8%,n=5), 加样回收率为99.1%（RSD1.9%,n=5）,检测限为3ng,该方法准确、方便、快速。     

Micro-magnetic particles frit for capillary electrochromatography

This paper presents a new method for making frit using soft-ferrite-based micro-magnetic particles (MMPs) in a micro-space, such as in a capillary tube. The MMPs-frit was made by injecting an aliquot of 10 microm (outer diameter; o.d.)-MMPs-suspension in methanol (ca. 1mg/ml) into a capillary tube (75 microm inner diameter (i.d.) x 375 microm o.d. x ca. 35 cm length) that was already sandwiched between a pair of cylindrical Neodium (Nd-Fe-B) magnets (1.5 mm o.d. x 1.5 mm height, 280 mT) at a position where the frit was made. The MMPs were trapped in the capillary tube as a frit due to the attraction of the magnets placed at surface on the capillary tube. With regard to durability, the frit was stable for methanol flow with a flow rate of 400 microl/min at room temperature. Using such a frit, a capillary column (20 cm long) was prepared by injecting a 5 microm (o.d.)-ODS-particle suspension in methanol (ca. 0.4 mg/microl) into the capillary tube. The MMPs-frits-ODS-packed column was stable for methanol for a flow pressure less than 20MPa. When comparing the present column with a conventional sintered-frits-ODS-packed column for the purposes of separating five kinds of biogenic amines by means of an on-column derivatization capillary electrochromatography (CEC), the performance of the MMPs-frits capillary column was almost equivalent to that of the sintered-frits-ODS-packed column.     

A validated liquid chromatographic/tandem mass spectrometric method for the determination of phencyclidine in microliter samples of rat serum

A liquid chromatographic/tandem mass spectrometric method is described for the determination of phencyclidine (PCP) in small volumes of rat serum (e.g. 50 microl). Samples were extracted using a mixed-mode strong cation-exchange column and then separated isocratically using a narrow-bore (2.1 mm i.d.) 3 microm Hypersil phenyl column and a mobile phase consisting of an ammonium formate buffer (pH 2.7) with 60% (v/v) methanol. Detection was accomplished using positive ion electrospray ionization in the multiple reaction monitoring mode. Mass spectra were obtained and peaks were observed at an m/z (% abundance) of 244 (100), 159 (25), and 86 (89). Tandem mass spectra were also obtained from the m/z 244 precursor ion with peaks observed at m/z 159 (100), 86 (96), and 91 (11). Optimum serum PCP sensitivity and precision were obtained at a transition of m/z 244 --> 159. Matrix-associated ion suppression did not significantly affect the accuracy (100-112%) or precision (CV < or =8%) of the assay. The lower limit of quantitation was 1 ng ml(-1) in 50 microl of serum. The method was used to study the serum pharmacokinetics of PCP in rats after an intravenous bolus dose of PCP.     

Development of an HPLC method for the monitoring of tricyclic antidepressants in biofluids

A simple, rapid and sensitive HPLC method was developed and validated for the determination of four tricyclic antidepressants (TCAs): amitriptyline, doxepin, clomipramine (CLO) and imipramine, in pharmaceutical formulations and biological fluids. A Kromasil C(8 )analytical column (250 x 4 mm, 5 microm) was used for the separation, with a mobile phase consisting of 0.05 M CH(3)COONH(4) and CH(3)CN (45:55 v/v) delivered at 1.5 mL/min isocratically. Quantification was performed at 238 nm, with bromazepam (1.5 ng/microL) as the internal standard. The determination of TCAs in blood plasma was performed after protein precipitation. Urine analysis was performed by means of SPE using Lichrolut RP-18 Merck cartridges providing high absolute recoveries (> 94%). Direct analysis of urine was also performed after two-fold dilution. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between-day precision (n = 5) revealed RSD <13%. Recoveries from biological samples ranged from 91.0 to 114.0%. The absolute detection limit of the method was calculated as 0.1-0.6 ng in blood plasma and 0.2-0.5 ng in extracted urine or 0.4-0.7 in diluted urine. The method was applied to real samples of plasma from a patient under CLO treatment.