Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

Preparation of biological fluids for simultaneous analysis of prostaglandin cyclo-oxygenase synthesized compounds by gas chromatography with electron capture detection

A method for isolating climax products of the arachidonic acid cascade from biological fluids is described which allows simultaneous measurement of PGs (PGE2, PGD2, 6-keto-PGF1 alpha, TXB2, 6-keto-PGE1, 6, 15-diketo-13,14-dihydro-PGF1 alpha) by electron capture detection of pentafluorobenzyloxime methyl ester trimethylsilyl (TMS) ether derivatives. PGs are adsorbed onto Amberlite XAD-2 from acidified solution and nonadhering substances removed by sequential administration of water, then petroleum ether. PGs are extracted into methanol. Following evaporation and reconstitution in water, the PGs are extracted into ethyl acetate at pH 3 and the ethyl acetate extracts are purified by lipidex column chromatography. Derivatization to pentafluorobenzyloxime methyl ester TMS ethers of PGs in the sample is followed by separation on either glass packed-columns or SCOT capillary columns, and detection by an electron capture detector. PGA2, added to the unpurified sample, is used as an internal standard for quantification. The methods have performed well on all biological fluids thus far examined. Examples of chromatographs from urine, Krebs-perfused lung effluents, and blood are shown.     

Determination of the active metabolite of sibutramine by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific method for the determination of the active primary amine metabolite of sibutramine, N-di-desmethylsibutramine (BTS 54,505), in human plasma was developed, based on high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS). The samples were extracted from plasma with methyl tert.-butyl ether, followed by separation and evaporation after addition of the internal standard, propranolol, and basification with sodium hydroxide. The residue was reconstituted in mobile phase and injected into the HPLC-MS-MS system. Chromatography was performed on an ODS MS column with a mobile phase consisting of acetonitrile (containing 0.1% trifluoroacetic acid, v/v)-0.1% trifluoroacetic acid (55:45, v/v) at a flow-rate of 0.3 ml/min. Multiple reaction monitoring using precursor-->product ion combinations at m/z 252.00-->125.00 and 260.00-->115.70 was applied to determine BTS 54,505 and propranolol, respectively. Linearity was confirmed in the concentration range 0.328-32.8 ng/ml in human plasma and the imprecision of this assay was less than 19.90% over the entire concentration range. The method is sufficiently sensitive and repeatable to be used in pharmacokinetic studies.     

Sensitive column-switching high-performance liquid chromatography method for determination of propiverine in human plasma

A sensitive column-switching high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of propiverine in human plasma. Propiverine and internal standard, oxybutynin, were extracted from human plasma that had been made basic with 5N sodium hydroxide into methyl tert-butyl ether. The extracted plasma sample was injected onto the HPLC system consisting of a pretreatment column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The assay was linear in concentration ranges of 2-200 ng/ml for propiverine in human plasma. This method showed excellent sensitivity (a limit of detection of 0.5 ng/ml), good precision and accuracy. This method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Rapid microdetermination of fatty acids in biological materials by gas-liquid chromatography

Total and free fatty acids in general ranging from lauric to nervonic acid were separated and quantitated based on an internal standard method as methyl esters by “on column” methylation with trimethyl-(α,α,α-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH) in a gas chromatographic system. This study represents an application of a method published by MacGee and Allen and a change to an internal standard technique. For the determination of the total fatty acids the sampls were saponified with KOH-CH₃OH, acidified with H₂PO₄, and then the fatty acids were extracted into hexane. An aliquot of the hexane extract was then extracted with TMTFTH and chromatographed. For determination of free fatty acids the sample was acidified with H₃PO₄, immediately extracted with hexane and processed as described earlier. The relative standard deviation of 1.4 to 4.2% illustrates the precision of the method and the recovery of the fatty acids ranged from 88.5 to 100.5%. This method was applied to the determination of fecal fatty acids in conjunction with an interdepartmental study on “High protein diet in colon cancer” at the University of Missouri. In addition, the applicability of the analytical procedure (with small modifications) was shown for a wide variety of biological materials (serum, milk, skin tissue, fungal spores, food homogenates, beef tissues, and tumor cell cultures). The analyses were performed on different gas chromatographs by different analysts.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.     

Determination by liquid chromatography-mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation

A rapid, sensitive and selective LC-MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C(18) analytical column, and a mobile phase of methanol-water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1ml/min. The limits of determination were 35pg/ml and 7pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.     

Efflux of dipicolinic acid from human erythrocytes, sealed membrane fragments, and band 3-liposome complexes: a fluorescence probe for the erythrocyte anion transporter

The greatly enhanced fluorescence of Tb3+ when complexed with dipicolinic acid affords a simple and highly sensitive method for monitoring continuous anion flux through the erythrocyte anion transporter, band 3. Dipicolinic acid (pyridine-2,6-dicarboxylic acid) is entrapped in human erythrocytes and other band 3-membrane preparations. Efflux of dipicolinic acid from preequilibrated systems into Tb3+-containing medium is monitored fluorometrically. Dipicolinic acid efflux is demonstrated in intact erythrocytes, sealed red cell membrane fragments, and band 3-liposome complexes. In each system, dipicolinic acid efflux is blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The technique is highly sensitive and reproducible, and is applicable to hemoglobin-containing and hemoglobin-free systems of widely varying surface area to volume ratios.     

Quantitative determination of BMS-186716, a thiol compound, in rat plasma by high-performance liquid chromatography-positive ion electrospray mass spectrometry after hydrolysis of the methyl acrylate adduct by the native esterases

During method development in support of non-clinical studies in animal models, BMS-186716 was found to be extremely unstable in blood and plasma. Stabilization of the compound was achieved by reacting the compound with methyl acrylate (MA) in blood, from which the plasma was then prepared. While the resulting BMS-186716-MA adduct was found to be stable in dog plasma, and hence it was used as the basis for the method developed for analysis of dog plasma samples, the BMS-186716-MA adduct was found to be unstable in rat plasma as it was readily hydrolyzed to BMS-186716-acrylic acid (AA) by native esterases found in rat plasma. Although the finding of the instability of BMS-186716-MA in rat plasma was not the result of prospective planning, we were able to successfully develop a quantitative bioanalytical method using BMS-186716-AA as the analyte instead of the originally planned BMS-186716-MA analyte. The standard and quality-control (QC) samples were prepared by spiking blank plasma with BMS-186716-MA, and then allowing them to stand at room temperature for 1 h to convert BMS-186716-MA to BMS-186716-AA. After adding the internal standard BMS-188035-AA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. The reconstituted extract was injected into a HPLC-electrospray ionization mass spectrometric system for detection by positive ion electrospray ionization. A lower limit of quantitation (LLQ) of 5 ng/ml was achieved, using 0.1 ml plasma and a standard curve range of 5–5000 ng/ml.     

Sensitive and selective method for the determination of chlormezanone in plasma by electron-capture gas chromatography

A sensitive and selective determination method of chlormezanone in plasma has been divised. Chlormezanone in plasma was extracted with toluene at pH 4.5, and converted into p-chlorobenzaldehyde in 0.1 N NaOH. Using p-bromobenzaldehyde as an internal standard, the hydrolysis product and the internal standard were extracted with n-hexane, and the extract was concentrated in vacuo in the presence of isoamyl alcohol to prepare the sample solution. The sample solution was submitted to electron-capture gas chromatography. Chlormezanone was determined by use of the peak height ratio of p-chlorobenzaldehyde against the internal standard. The method was utilized successfully for pharmacokinetic studies of chlormezanone in plasma.     

Rapid and sensitive determination of paclitaxel in mouse plasma by high-performance liquid chromatography

This report describes a rapid, simple and sensitive isocratic high-performance liquid chromatography with diode array UV detection for micro-sample analysis of paclitaxel in mouse plasma. The analysis utilized a Capcell-pak octadecyl analytical column and a mobile phase consisting of acetonitrile–0.1% phosphoric acid in deionized water (55:45, v/v). Paclitaxel and n-hexyl p-hydroxybenzoic acid (internal standard) were extracted from plasma by one-step extraction with tert.-butyl methyl ether. Peak purity was determined over a UV wavelength range of 200 to 400 nm. Paclitaxel and the internal standard were eluted at 3.4 min and 5.4 min, respectively, at a mobile phase flow-rate of 1.3 ml/min. No interfering peaks were observed and the total run time was 10 min. The standard curve was linear (r=0.9999) over the concentration range of 0.010–500 μg/ml. The extraction recovery was >90% for both paclitaxel and n-hexyl p-hydroxybenzoic acid. The intra- and inter-day assay variabilities of paclitaxel ranged from 0.4 to 2.2% and 0.6 to 7.8%, respectively. The LOD and LOQ were 5 and 10 ng/ml, respectively, for paclitaxel using a plasma sample volume of 100 μl. This highly sensitive and simple assay method was successfully applied to a pharmacokinetic study after i.v. administration of paclitaxel 20 mg/kg to mice.     

Stereoselective determination of R-(+)- and S-(−)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(−)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30–32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 μg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.     

Measurement of paclitaxel in biological matrices: high-throughput liquid chromatographic-tandem mass spectrometric quantification of paclitaxel and metabolites in human and dog plasma

A GLP-validated, sensitive and specific LC-MS-MS method for the quantification of paclitaxel and its 6-alpha- and 3'-p-hydroxy metabolites is presented. A 0.400 ml plasma aliquot is spiked with a (13)C(6)-labeled paclitaxel internal standard and extracted with 1 ml methyl-tert.-butyl ether. The ether is evaporated and the residue is reconstituted in 130 microl of 30% aqueous acetonitrile (ACN) containing 0.1% trifluoroacetic acid. Isocratic HPLC analysis is performed by injecting 50 microl of the reconstituted material onto a 50x2.1 mm C(18) column with an ACN-water-acetic acid (50:50:0.1) mobile phase at 200 microl/min flow. Detection is by positive ion electrospray followed by multiple reaction monitoring of the following transitions: paclitaxel (854>509 u), 6-alpha-hydroxy paclitaxel (870>525 u), 3'-p-hydroxy paclitaxel (870>509 u) and internal standard (860>509 u). Quantification is by peak area ratio against the 13C(6) internal standard. The method range is 0.117-117 nM (0.1-100 ng/ml) for paclitaxel and both metabolites using a 0.400 ml human or dog plasma sample. Analysis time per sample is less than 5 min.     

Quantitation of the fatty acid composition of phosphatidic acid by capillary gas chromatography electron-capture detection with picomole sensitivity

We describe a relatively simple and sensitive method to measure fentomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in sensitivity compared to the most sensitive of the previously described methods. An example is provided of quantitating phosphatidic acid from minute amounts of biological samples such as islets of Langerhans.     

Quantification of lipoic acid in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of lipoic acid (LA) in human plasma. LA and the internal standard, naproxen, were extracted from a 500 microl plasma sample by one-step deproteination using acetonitrile. Chromatographic separation was performed on a Zorbax SB-C(18) Column (100 mmx3.0mm i.d. with 3.5 microm particle size) with the mobile phase consisting of acetonitrile and 0.1% acetic acid (pH 4, adjusted with ammonia solution) (65:35, v/v), and the flow rate was set at 0.3 ml/min. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was linear over the concentration range of 5-10,000 ng/ml for LA. The intra- and inter-day precisions were less than 7% and accuracy ranged from -7.87 to 9.74% at the LA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around time. The method has been successfully applied to a clinical pharmacokinetic study of LA in 10 healthy subjects.     

Determination of metformin in human plasma by high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of antihyperglycemic agent metformin in human plasma using a novel sample extraction procedure. Liquid-liquid extraction of metformin and ranitidine (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (50:50, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a silica column (250 mmx4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (25:75, v/v), pH 6. The limit of quantification (LOQ) was 15.6 ng/ml and the calibration curves were linear up to 2000 ng/ml. The mean absolute recoveries for metformin and internal standard using the present extraction procedure were 98 and 95%, respectively. The intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 8.3%.     

Determination of opipramol in human plasma by high-performance liquid chromatography with photometric detection using a cyanopropyl column

A high-performance liquid chromatographic method is described for the determination of opipramol in human plasma. Opipramol was extracted into tert.-butylmethyl ether, separated on a cyanopropyl silica column and detected at 254 nm. Imipramine was used as internal standard. The limit of quantitation was 250 pg/ml using 1.5 ml plasma. Precision was better than 9%, inaccuracy less than 8%. The assay is more sensitive than previously published methods, and it has been applied to the analysis of plasma samples from a pharmacokinetic study.     

Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.     

气相色谱法测定抗毒素中的间甲酚含量

采用气相色谱法测定抗毒素中的间甲酚含量。样品用苯甲醇作内标,经高氯酸沉淀蛋白、氯仿抽提后,通过HP-INNO-WAX柱,以氢火焰检测器测定间甲酚含量。内标峰与样品峰的分离度大于1.5,平均回收率及变异系数分别为100.067%和1.93%,测定线性范围0.0202~1.7588mg/ml,最小检测限为0.01272mg/ml,该方法准确快速,比化学方法好。     

Determination of ambroxol in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI)

A rapid, sensitive and specific method to determination of ambroxol in human plasma using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/ESI) was described. Ambroxol and the internal standard (I.S.), fentanyl, were extracted from plasma by N-hexane-diethyl ether (1:1, v/v) after alkalinized with ammonia water. A centrifuged upper layer was then evaporated and reconstituted with 100 microl mobile phase. Chromatographic separation was performed on a BDS HYPERSIL C18 column (250 mmx4.6 mm, 5.0 microm, Thermo electron corporation, USA) with the mobile phase consisting of 30 mM ammonium acetate (0.4% formic acid)-acetonitrile (64:36, v/v) at a flow-rate of 1.2 mL min(-1). The total run time was 5.8 min for each sample. Detection and quantitation was performed by the mass spectrometer using selected ion monitoring at m/z 261.9, 263.8 and 265.9 for ambroxol and m/z 337.3 for fentanyl. The calibration curve was linear within the concentration range of 1.0-100.0 ng mL(-1) (r=0.9996). The limit of quantification was 1.0 ng mL(-1). The extraction recovery was above 83.3%. The methodology recovery was higher than 93.8%. The intra- and inter-day precisions were less than 6.0%. The method is accurate, sensitive and simple for the study of the pharmacokinetics and metabolism of ambroxol.