Rapid analysis of tetracaine for a tape stripping pharmacokinetic study using short‐end capillary electrophoresis

A rapid and simple short‐end (reverse) capillary zone electrophoresis method was developed and validated for the separation and quantification of tetracaine in skin using tape samples. The separation was performed in a 485 mm (400 mm to window) × 50 µm internal diameter fused silica capillary using a background electrolyte of phosphoric acid–Tris pH2.5 at –25 kV. The extraction of tetracaine from tape samples was achieved using methanol diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and tetracaine were 1.25 and 1.36 min, respectively. The limit of quantification for tetracaine was 50 µg, with a signal‐to‐noise ratio greater than 10. The calibration curve was linear from 50 to 1200 µg with r² greater than 0.99. The CV for both within‐ and between‐assay imprecision and the percentage inaccuracy for the quality control samples including lower and upper limits of quantitation were <12.1% and <11%, respectively. The absolute mean recovery of tetracaine was >97%. The accuracy and selectivity of this method allowed the rapid measurement of tetracaine in tape samples obtained from a skin tape stripping study of local anaesthetics in healthy subjects. Copyright © 2008 John Wiley & Sons, Ltd.     

Continuous capillary electrophoresis with flow injection and its application for determination of Ephedrine and Pseudo-ephedrine in Chinese medicinal preparations

This article presents a new, simple and rapid continuous separation method by combination of flow injection with capillary electrophoresis designed for the analysis of basic traditional Chinese medicines. The device was produced using commercial capillary and components readily available in analytical laboratory. In double-T configuration, the designed horizontal separation channel was 25 microm i.d. x 146 mm length (an effective separation length of 93 mm) quartz capillary, with two vertical elicitation arms produced from 0.5 mm i.d. pump tubing. The capillary was embedded in a 40 x 20 x 3 mm organic glass base. Using the double-T configuration, continuous introduction of a series of samples was achieved. More than 3.00 resolution for ephedrine and pseudo-ephedrine were obtained using 100 mm borate buffer (pH 9.80) within 8 min in 25 microm separation channel with an electrical field strength of 137 V/cm (UV detection at 215 nm). The linear calibration range was 50-1500 microg/mL (ephedrine, r = 0.9982; pseudo-ephedrine, r = 0.9990) for both analytes. The limits of detection were 2.65 micro g/mL for ephedrine and 2.92 microg/mL for pseudo-ephedrine. In this device, the contents of ephedrine and pseudo-ephedrine in five Chinese medicinal preparations were determined with RSDs (n = 5) in range 1.16-4.51% and recoveries in range 90.4-114.6%.     

Stability-indicating HPLC method for acyclovir and lidocaine in topical formulations

A simple, rapid and accurate stability-indicating HPLC assay was developed for the determination of acyclovir and lidocaine in topical formulations. Chromatographic separation of acyclovir and lidocaine was achieved using a reversed-phase C₁₈ column and a gradient mobile phase (20 mm ammonium acetate pH 3.5 in water and acetonitrile). The degradation products of acyclovir and lidocaine in the samples were analyzed by ultra performance liquid chromatography-time of flight mass spectrometry. The HPLC method successfully resolved the analytes from the impurities and degradation products in the topical formulation. Furthermore, the method detected the analytes from the human skin leachables following the extraction of the analytes in the skin homogenate samples. The method showed linearity over wide ranges of 5–500 and 10–200 μg/ml for acyclovir and lidocaine in the topical product, respectively, with a correlation coefficient (r²) >0.9995. The relative standard deviations for precision, repeatability, and robustness of the method validation assays were <2%. The skin extraction efficiency for acyclovir and lidocaine was 92.8 ± 0.7% and 91.3 ± 3.2%, respectively, with no interference from the skin leachables. Thus, simultaneous quantification of acyclovir and lidocaine in the topical formulations was achieved.     

Surfactant‐assisted dispersive liquid–liquid microextraction combined with field‐amplified sample stacking in capillary electrophoresis for the determination of mexiletine and lidocaine

A sensitive method for the determination of mexiletine and lidocaine using surfactant‐assisted dispersive liquid–liquid microextraction coupled with capillary electrophoresis was developed. Triton X‐100 and dichloromethane were used as the dispersive agent and extraction solvent, respectively. After the extraction, mexiletine and lidocaine were analyzed using capillary electrophoresis with ultraviolet detection. The detection sensitivity was further enhanced through the use of field‐amplified sample stacking. Under optimal extraction and stacking conditions, the calibration curves were linear over a concentration range of 0.05–1.00 μM for mexiletine and 0.03–1.00 μM for lidocaine. The limits of detection (signal‐to‐noise ratio of 3) were 0.01 and 0.01 μM for mexiletine and lidocaine, respectively. An approximately 1141‐ to 1250‐fold improvement in sensitivity was observed for the two analytes compared with the injection of a standard solution without the surfactant‐assisted dispersive liquid–liquid microextraction and field‐amplified sample stacking procedures. This developed method was successfully applied to the determination of mexiletine and lidocaine in human urine and serum samples. Both precision and accuracy for urine and serum samples were less than 8.7 and 6.7%, respectively. The recoveries of the two analytes from urine and serum samples were 54.7–64.9% and 16.1–56.5%, respectively.     

Determination of scopolamine, atropine and anisodamine in Flos daturae by capillary electrophoresis.

A capillary electrophoresis method was developed for the separation and determination of tropane alkaloids in Flos daturae plants. Separation was performed on a fused silica capillary(42.1 cm x 50 microm i.d.) at an applied voltage of 20 kV. Scopolamine, atropine and anisodamine were well separated in the buffer of 50 mmol/L phosphate buffer (pH 5.0) containing 20% (v/v) tetrahydrofuran (THF). Beer's law was obeyed in the range of concentration of 2.4-21.8 microg/mL for scopolamine, 4.0-36.0 microg/mL for atropine and 2.6-23.7 microg/mL for anisodamine, respectively, and the correlation coefficients were over 0.999 (n = 6). The developed method was applied for the analysis of herb samples.     

Separation and determination of ephedrine and pseudoephedrine by combination of flow injection with capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of ephedrine and pseudoephedrine using direct UV absorbance detection has been developed by the combination of flow injection with capillary electrophoresis for the first time. The buffer solution used is a 40 mM borate solution with the pH adjusted to 9.5 using a 2 M NaOH solution. The linear calibration range is 50 to 1000 microg/mL (r = 0.9996) for both analytes, and the recoveries are 91.2-108.2% for ephedrine and 92.6-107.3% for pseudoephedrine, respectively. The relative standard deviation of the peak area is 1.6% for ephedrine and 1.3% for pseudoephedrine (n = 6) at a concentration of 500 microg/mL, respectively. A series of samples is injected repeatedly without current interruption and subsequent rinsing, and the contents of these two alkaloids in three marketed drugs and the medical plant, Ephedra sinica, are determined with satisfactory results by this method.     

Determination of low molecular weight alcohols including fusel oil in various samples by diethyl ether extraction and capillary gas chromatography

Low molecular weight alcohols including fusel oil were determined using diethyl ether extraction and capillary gas chromatography. Twelve kinds of alcohols were successfully resolved on the HP-FFAP (polyethylene glycol) capillary column. The diethyl ether extraction method was very useful for the analysis of alcohols in alcoholic beverages and biological samples with excellent cleanliness of the resulting chromatograms and high sensitivity compared to the direct injection method. Calibration graphs for all standard alcohols showed good linearity in the concentration range used, 0.001-2% (w/v) for all alcohols. Salting out effects were significant (p < 0.01) for the low molecular weight alcohols methanol, isopropanol, propanol, 2-butanol, n-butanol and ethanol, but not for the relatively high molecular weight alcohols amyl alcohol, isoamyl alcohol, and heptanol. The coefficients of variation of the relative molar responses were less than 5% for all of the alcohols. The limits of detection and quantitation were 1-5 and 10-60 microg/L for the diethyl ether extraction method, and 10-50 and 100-350 microg/L for the direct injection method, respectively. The retention times and relative retention times of standard alcohols were significantly shifted in the direct injection method when the injection volumes were changed, even with the same analysis conditions, but they were not influenced in the diethyl ether extraction method. The recoveries by the diethyl ether extraction method were greater than 95% for all samples and greater than 97% for biological samples.     

Determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine-5'-monophosphate in pancreatic cancer cell line and other biological materials using capillary electrophoresis

Capillary zone electrophoresis (CZE) was used for the rapid determination of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in pancreatic cancer cell line (PANC-1), culture medium, plasma and pancreatic tissue. The assay is based upon protein precipitation with acetonitrile followed by a 9-min CZE analysis of the supernatant in an uncoated fused-silica capillary employing a borate buffer and on-column absorbance detection at 265 nm. Using 50 microl of sample, 5-FU levels between 4.12 and 132 microg/ml (31.7-1000 microM) were found to provide linear calibration graphs. Intra-day and inter-day RSD values evaluated from peak height ratios (n=5) were <7.6 and <8.8%, respectively. Corresponding RSD values of detection times (n=7) were <1 and <1.5%, respectively. The limits of detection for 5-FU and FdUMP were 1.72 and 5.29 microg/ml, respectively. As application, the accumulation of 5-FU by PANC-1 cells over a 4-h time period was investigated. Having a culture medium concentration of 100 microg/ml, the 5-FU cell content was estimated to become equal to that of the surrounding medium (i.e., 100 microg/ml or 3.61 fmol per cell with a volume of 4.7 pl) within that time period. The sensitivity of the assay was sufficient for the determination of 5-FU in all cell samples. FdUMP, however, could not be detected in these samples. Furthermore, the data obtained in uncoated capillaries are compared to those measured in a fused-silica capillary whose inner surface was coated with linear polyacrylamide (about 10-fold reduction of electroosmosis). The latter capillary format was found to be useless for simultaneous analysis of 5-FU and FdUMP in pancreatic cells but could be potentially useful for analysis of these compounds in plasma.     

一滴溶剂微萃取-毛细管气相色谱法分析水中的七种硝基苯类化合物

用一滴溶剂微萃取(SDME)-毛细管气相色谱联用技术测定水中的硝基苯、硝基甲苯类和硝基氯苯类化合物,对影响萃取的因素如萃取溶剂种类、液滴体积、搅拌速度、针尖入水深度、水样体积、萃取时间、萃取温度等进行了优化,结果表明:硝基苯和硝基甲苯类化合物在0.8～32 μg/L 范围内,硝基氯苯类化合物在0.04～3.2 μg/L 范围内均呈现良好的线性(r2>0.999),检出限可达0.01～0.3 μg/L。自来水加标样品测定的相对标准偏差和平均回收率(n=5)范围分别为3.1%～7.9%和101%～105%,废水加标样品测定的相对标准偏差和平均回收率(n=5)范围分别为3.3%～7.9%和92.5%～97.0%。优化后的SDME具有环保、灵敏、快速、简便等特点,适用于萃取水中的痕量硝基苯、硝基甲苯类和硝基氯苯类化合物。     

Rapid determination of cyclamate in foods by solid-phase extraction and capillary electrophoresis

A method for the determination of cyclamate in food was developed using solid-phase extraction (SPE) and capillary electrophoresis (CE) with indirect ultraviolet (UV) detection. A 5-10 g sample in 0.1 mol/L hydrochloric acid was homogenized and made up to a volume of 50 mL with 0.1 mol/L hydrochloric acid. After the sample was centrifuged, 25 mL of supernatant was loaded into an Oasis HLB SPE cartridge. The cartridge was washed with 2 mL of demineralized water followed by 2 mL of 50% aqueous methanol, and cyclamate was eluted with 4.5 mL of 50% aqueous methanol. The eluate was added to a solution of sodium propionate (internal standard) for CE analysis. The cyclamate in the eluate was electrophoresed on a fused-silica capillary using 1 mmol/L hexadecyltrimethylammonium bromide and 10 mmol/L potassium sorbate as a running buffer. Detection and reference wavelengths of cyclamate determined with a UV detector were 300 and 254 nm, respectively. The calibration curves for cyclamate showed good linearity in the range of 2-1000 microg/mL and the limits of detection in beverage, fruit in syrup, jam, pickles and confectionary are sample dependent and ranged from 5-10 microg/g. The recovery of cyclamate added at a level of 200 microg/g to various kinds of foods was 93.3-108.3% and the relative standard deviation was less than 4.9% (n=3). A number of commercial samples were analyzed using the proposed method. Cyclamate was detected in one waume, two pickles, and two sunflower seeds. The quantitative values determined with CE correlated to those from high-performance liquid chromatography (HPLC) (the detected values of cyclamate in a sunflower seed measured by CE and HPLC were 3.40 g/kg and 3.51 g/kg, respectively). This analytical method for cyclamate using CE is especially suitable for use in the field.     

Determination of iodide in samples with complex matrices by hyphenation of capillary isotachophoresis and zone electrophoresis

A method has been developed for the determination of iodide in mineral water, seawater, cooking salt, serum, and urine based on hyphenation of capillary ITP and zone electrophoresis. A commercially available instrumentation for capillary ITP with column-switching system was used. ITP served for removal of chloride present in the analyzed samples in a ratio of 10(6)-10(7):1 to iodide, zone electrophoresis was used for evaluation. Isotachophoretic separation proceeded in a capillary made of fluorinated ethylene-propylene copolymer of 0.8 mm id and 90 mm total length to the bifurcation point filled with a leading electrolyte (LE) composed of 8 mM HCl + 16 mM beta-alanine (beta-Ala) + 10% PVP + 2.86 mM N(2)H(4)x2HCl, pH 3.2; and a terminating electrolyte composed of 8 mM H(3)PO(4) + 16 mM beta-Ala + 10% PVP + 5 mM N(2)H(4), pH 3.85 for all the matrices except seawater. For ITP of seawater the LE consisted of 50 mM HCl + 100 mM beta-Ala + 10% PVP + 2.86 mM N(2)H(4)x2HCl, pH 3.52. Distance of conductivity detector from the injection point and bifurcation point was 52 and 38 mm, respectively. Zone electrophoresis was performed in a capillary made of fused silica of 0.3 mm id and 160 mm total length filled with LE from isotachophoretic step. LODs reached for all matrices were 2-3x10(-8) M concentration (2.5-4 microg/L) enabled monitoring of iodide in all analyzed samples with RSD 0.4-9.3%. Estimated concentrations of iodide in individual matrices were 10(-6)-10(-8) M.     

Capillary electrophoresis on-line coupled with hydride generation-atomic fluorescence spectrometry for speciation analysis of selenium

A new method for speciation analysis of two inorganic selenium species was developed by on-line coupling of capillary electrophoresis (CE) with hydride generation-atomic fluorescence spectrometry (HG-AFS) and on-line conversion of Se(VI) to Se(IV). Baseline separation of Se(VI) and Se(IV) was achieved by CE in a 50 cm x 75 microm inside diameter (ID) fused-silica capillary at -20 kV using a mixture of 15 mmol.L(-1) NaH2PO4 and 0.5 mmol.L(-1) cetyltrimethylammonium bromide (pH 7.5) as electrolyte buffer. Se(VI) was on-line reduced to Se(IV) by mixing the CE effluent with concentrated HCl. The precision (relative standard deviation, RSD, n=7) ranged from 0.7 to 1.3% for migration time, 6.4 to 3.7% for peak height response, and 5.9 to 6.1% for peak area for the two selenium species at the 500 microg.L(-1) (as Se) level. The detection limits were 33 and 25 microg.L(-1) (as Se) for Se(VI) and Se(IV), respectively. The recoveries of the two selenium species in five locally collected water samples ranged from 88 to 114%. The developed method was applied to speciation analysis of inorganic selenium species in spiked natural water samples.     

Validation of high-performance liquid chromatographic-mass spectrometric method for the analysis of lidocaine in human plasma

A sensitive and simple liquid chromatography-tandem mass spectrometry method is developed and validated for the determination of lidocaine in human plasma. Bupivacaine is used as an internal standard, and the plasma extraction is performed by a simple liquid-liquid extraction. The limit of quantitation (LOQ) is 0.5 ng/mL with a signal-to-noise ratio greater than 5. The calibration curve is linear from 0.5 to 250 ng/mL with an r2 greater than 0.99. The coefficients of variation for within- and between-assay imprecision, including LOQ, are < or = 13% and < or = 8%, respectively. The percentage of inaccuracy for within- and between-assay, including LOQ, are < or = 9% and < or = 5%, respectively. The absolute recovery of lidocaine and bupivacaine are greater than 84% and 82%, respectively. The higher sensitivity and accuracy of this method allow the measurement of low concentrations of lidocaine in plasma from a clinical study of topically applied lidocaine in healthy subjects.     

Semi-permeable surface analytical reversed-phase column for the improved trace analysis of acidic pesticides in water with coupled-column reversed-phase liquid chromatography with UV detection. Determination of bromoxynil and bentazone in surface water.

The coupled-column (LC-LC) configuration consisting of a 3 microm C18 column (50 x 4.6 mm I.D.) as the first column and a 5 microm C18 semi-permeable-surface (SPS) column (150 x 4.6 mm I.D.) as the second column appeared to be successful for the screening of acidic pesticides in surface water samples. In comparison to LC-LC employing two C18 columns, the combination of C18/SPS-C18 significantly decreased the baseline deviation caused by the hump of the co-extracted humic substances when using UV detection (217 nm). The developed LC-LC procedure allowed the simultaneous determination of the target analytes bentazone and bromoxynil in uncleaned extracts of surface water samples to a level of 0.05 microg/l in less than 15 min. In combination with a simple solid-phase extraction step (200 ml of water on a 500 mg C18-bonded silica) the analytical procedure provides a high sample throughput. During a period of about five months more than 200 ditch-water samples originating from agricultural locations were analyzed with the developed procedure. Validation of the method was performed by randomly analyzing recoveries of water samples spiked at levels of 0.1 microg/l (n=10), 0.5 microg/l (n=7) and 2.5 microg/l (n=4). Weighted regression of the recovery data showed that the method provides overall recoveries of 95 and 100% for bentazone and bromoxynil, respectively, with corresponding intra-laboratory reproducibilities of 10 and 11%, respectively. Confirmation of the analytes in part of the samples extracts was carried out with GC-negative ion chemical ionization MS involving a derivatization step with bis(trifluoromethyl)benzyl bromide. No false negatives or positives were observed.     

Fabrication of microfluidic devices using dry film photoresist for microchip capillary electrophoresis

An inexpensive, disposable microfluidic device was fabricated from a dry film photoresist using a combination of photolithographic and hot roll lamination techniques. A microfluidic flow pattern was prefabricated in a dry film photoresist tape using traditional photolithographic methods. This tape became bonded to a poly(methyl methacrylate) (PMMA) sheet with prepouched holes when passed through a hot roll laminator. A copper working electrode and platinum decoupler was readily incorporated within this microchip. The integrated microchip device was then fixed in a laboratory-built Plexiglas holder prior to its use in microchip capillary electrophoresis. The performance of this device with amperometric detection for the separation of dopamine and catechol was examined. The separation was complete within 50 s at an applied potential of 200 V/cm. The relative standard deviations (RSD) of analyte migration times were less than 0.71%, and the theoretical plate numbers for dopamine and catechol were 3.2 x 10(4) and 4.1 x 10(4), respectively, based on a 65 mm separation channel.     

Development of a validated capillary electrophoresis method for enantiomeric purity testing of dexchlorpheniramine maleate

A capillary zone electrophoresis method has been developed for the detection of 0.1% of (R)-levochlorpheniramine maleate in samples of (S)-dexchlorpheniramine maleate. Using 1.5 mM carboxymethyl-beta-cyclodextrin in an acidic background electrolyte, resolution values of more than 10 were obtained. Under these conditions the R-enantiomer is migrating in front of the bulk S-enantiomer. The assay was validated for linearity (2-10 microg/ml; R2 = 0.9992), selectivity [(RS)-pheniramine maleate and (RS)-brompheniramine maleate], limit of detection (0.25 microg/ml), limit of quantification (0.75 microg/ml), analytical precision (intra- and inter-day variability), repeatability of the method (RSD = 5.0%) and accuracy. In samples of dexchlorpheniramine maleate from two different manufacturers, concentrations of, respectively, 0.15% and 1.95% (m/m) of levochlorpheniramine maleate were detected. The method was compared to the HPLC method described in the European Pharmacopoeia III monograph.     

Simultaneous quantitation of rosuvastatin and gemfibrozil in human plasma by high-performance liquid chromatography and its application to a pharmacokinetic study

A simple, sensitive and specific high-performance liquid chromatography method is described for simultaneous determination of rosuvastatin (RST) and gemfibrozil (GFZ) in human plasma using celecoxib as an internal standard (IS). The assay procedure involved extraction of RST, GFZ and IS from plasma into acetonitrile. Following separation and evaporation of the organic layer the residue was reconstituted in the mobile phase and injected onto an X-Terra C(18) column (4.6 x 150 mm, 5.0 microm). The chromatographic run time was less than 20 min using flow gradient (0.0-1.60 mL/min) with a mobile phase consisting of 0.01 M ammonium acetate:acetonitrile:methanol (50:40:10, v/v/v) and UV detection at 275 nm. Nominal retention times of RST, GFZ and IS were 6.7, 13.9 and 16.4 min, respectively. Absolute recovery of both analytes and IS was greater than 90%. The lower limit of quantification (LLOQ) of RST and GFZ was 0.03 and 0.30 microg/mL, respectively. Linearity was excellent (r(2) = 0.999) in the 0.03-10 microg/mL and 0.3-100 microg/mL ranges for RST and GFZ, respectively. The inter- and intra-day precisions in the measurement of RST quality control (QC) samples 0.03, 0.09, 2.50 and 8.00 microg/mL were in the range 2.37-9.78% relative standard deviation (RSD) and 0.92-10.08% RSD, respectively. Similarly, the inter- and intra-day precisions in the measurement of GFZ quality control (QC) samples 0.30, 0.90, 25.0 and 80.0 microg/mL were in the ranges 2.79-6.27 and 0.96-9.69% RSD, respectively. Accuracies in the measurement of QC samples for RST and GFZ were in the range 85.43-107.23 and 84.98-102.35% respectively, of the nominal values. RST and GFZ were stable in the array of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. Stability of RST and GFZ was established for 1 month at -80C. The application of the assay in an oral pharmacokinetic study in rats co-administered with RST and GFZ is described.     

Rapid target analysis for pesticides in water by online coated capillary microextraction combined with liquid chromatography and tandem mass spectrometry

The applicability of online trace enrichment with custom-made coated capillaries combined with tandem mass spectrometry was demonstrated for the target analysis of selected pesticides (mainly herbicides, e.g., triazines, phenylureas, and acetanilides) in water. The developed method allows rapid determination of several widely used plant protectants within a total analysis time of 11 min. Good linearity (r > or = 0.995) was obtained for the selected pesticides in the range of 0.050-50 microg/L. The relative standard deviations (RSDs) of the peak areas were < or = 3.8% for spiked Milli-Q water (5 microg/L). The RSDs obtained in analyses of spiked (1 microg/L) water samples (brook water, river water, sewage plant effluent) ranged from 2.9 to 6.8%, indicating low influence of the matrix on enrichment and detection. The detection limits, which ranged from 10 to 90 ng/L, fulfilled the requirements of the European regulations for drinking water. The polyacrylate coating of the extraction capillary showed good stability in the presence of water and acetonitrile and allowed > or = 100 extractions with 1 capillary.     

Separation of sympathomimetic amines of abuse and related compounds by micellar electrokinetic chromatography.

Separation of twelve sympathomimetic amines and related compounds by micellar electrokinetic chromatography (MEKC) with UV absorbance detection is described. These amines were well separated within 25 min using 50 mM sodium tetraborate solution containing 15 mM sodium dodecylsulfate (SDS) of pH 9.3 as a running solution and detected at 210 nm. MEKC was performed with an applied voltage of 13 kV at 25 degrees C using a fused-silica capillary (50 cm x 75 mm i.d.) with effective length of 37.5 cm. The detection limits of these compounds were in the range from 4 to 97 fmol/injection at a signal-to-noise ratio (S/N) of 3. The reproducibility of the method expressed as relative standard deviation (RSD) for within-day (n=6) and between-day (n=5) assays was less than 4.8 and 8.8%, respectively. The proposed method could be applied to the determination of an anorectic drug, phentermine, in Chinese tea with a detection limit of 99 microg/g (105 fmol/injection, S/N=3).     

Development and validation of a rapid headspace gas chromatography-mass spectrometry method for the determination of diethyl ether and acetone residues in Tween extracts of shellfish intended for mouse bioassay for diarrhoetic toxins

A validated analytical method using headspace capillary gas chromatography with mass spectrometric detection, which utilises deuterated analogues of the target analytes as internal standards has been developed and applied to the determination of acetone and diethyl ether in Tween extracts of cockles (Cerastoderma edule) and mussels (Mytilus edulis) destined for mouse bioassay for lipophilic toxins. The optimal conditions for headspace incubation were 50 degrees C for 6 min. The limits of detection and quantitation for both DEE and acetone were 2 and 7 microg/mL, respectively, based on signal to noise ratios of 3 and 10, respectively. The linear dynamic range of the instrument was 0 to ca. 4000 microg/mL for both acetone (r(2)=0.995) and DEE (r(2)=0.999). Tween extracts of cockle spiked with acetone and DEE at 3925 and 3570 microg/mL, respectively, gave mean (n=3) recovery figures of 101% (RSD=13.1%) for acetone and 90% (RSD=7.3%) for DEE in cockle matrix. The corresponding figures obtained from spiked mussel matrix were 114% (RSD=5.7%) for acetone and 95% (RSD=6.7%) for DEE, respectively, which were within acceptable range.