Chiral gas chromatographic assay with flame ionization detection for amphetamine enantiomers in microsomal incubates

A chiral assay for amphetamine enantiomers in rat liver microsomal incubates is based on derivatization with (S)-(-)-N-(trifluoroacetyl)-prolyl chloride (S-TFPC), capillary chromatographic separation of the diastereomeric amide derivatives, and detection by a flame ionization detector. The method is capable of detecting low levels of S- or R-amphetamine. The assay is linear from 5 to 250 micrograms/mL for each enantiomer, and the limit of detection is 0.5 microgram/mL. The analytical method affords the average recoveries of 77.53 +/- 5.22% for R-amphetamine and 74.47 +/- 3.08% for S-amphetamine. The method allows the study of the metabolic depletion of S- and R-amphetamine in rat liver microsomal incubates. The time-dependent concentration of amphetamine enantiomers in rat liver microsomes was determined, and the stereoselectivity of amphetamine phase I metabolism was observed.     

Stereoselective liquid chromatographic determination of 1'-oxobufuralol and 1'-hydroxybufuralol in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction (HF-LPME) method for the stereoselective determination of bufuralol metabolites 1'-oxobufuralol (1'-Oxo-BF) and 1'-hydroxybufuralol (1'-OH-BF) in microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H column with hexane/2-propanol/methanol (97.5:2.0:0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol/L acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63-69%. The method was linear over the concentration range of 100-5000 ng/mL for each enantiomer of 1'-Oxo-BF (r>0.9978) and of 100-2500 ng/mL for each stereoisomer of 1'-OH-BF (r>0.9957). The quantification limits were 100 ng/mL for all analytes. The validated method was used to assess the in vitro biotransformation of bufuralol using rat liver microsomal fraction that demonstrated predominant formation of (S)-1'-Oxo-BF and (R,R)-1'-OH-BF.     

Stereoselective high-performance liquid chromatographic assay for pirmenol enantiomers in dog plasma.

Pirmenol enantiomers in dog plasma were quantified using a stereospecific high-performance liquid chromatographic method with ultraviolet detection at 262 nm. Racemic pirmenol and internal standard, (+)-propranolol, were isolated from dog plasma by a three-step extraction procedure using toluene, 0.1 M hydrochloric acid and hexane, respectively. A chiral analytical column (Chiralcel OJ) was used with a mobile phase consisting of hexane-isopropanol-diethylamine (98.9:1.0:0.1). Linear calibration curves were obtained in the concentration range 0.0200-5.00 micrograms/ml for each enantiomer. Precision of the method, expressed as coefficient of variation for nine quality control samples, was 7.1% for (+)-pirmenol and 6.4% for (-)-pirmenol. Bias was +/- 2.2% for (+)-pirmenol and +/- 1.5% for (-)-pirmenol in quality control samples.     

HPLC analysis and pharmacokinetics of icariin in rats

As a prerequisite to the determination of pharmacokinetic parameters of icariin in rats, an HPLC method using UV detection was developed and validated. Icariin and the internal standard, quercetin, were extracted from plasma samples using ethyl acetate after acidification with 0.05 mol/L NaH2PO4 solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB Cls column (250 x 4.6 mm id, 5 microm) equipped with a Shim-pack GVP-ODS C18 guard column (10 x 4.6 mm id, 5 microm) using a mobile phase of ACN/water/acetic acid (31:69:0.4 v/v/v) at a flow rate of 1.0 mL/ min. Detection was at 277 nm. The calibration curve was linear from 0.05 to 100.0 microg/mL with 0.05 microg/mL as the lower LOQ (LLOQ) in plasma. The intra- and interday precisions in terms of RSD were lower than 5.7 and 7.8% in rat plasma, respectively. The accuracy in terms of relative error (RE) ranged from -1.6 to 3.2%. The extraction recoveries of icariin and quercetin were 87.6 and 80.1%, respectively. The main pharmacokinetic parameters for rats were determined after a single intravenous administration of 10 mg/kg icariin: t1/2, 0.562 +/- 0.200 h; AUC0-infinity, 8.73 +/- 2.23 microg x h/mL; CLToT, 20.10 +/- 5.80 L/kg x h; Vz, 1.037 +/- 0.631 L/kg; MRT0-infinity, 0.134 +/- 0.040 h; and Vss, 0.170 +/- 0.097 L/kg.     

反相高效液相色谱法测定人血浆中利培酮及其代谢物的质量浓度

建立了测定人血浆中利培酮及其活性代谢物9-羟利培酮质量浓度的反相高效液相色谱方法。用Zor－baxODSC18色谱柱,以V（甲醇）：V（水）：V（1mol／L醋酸铵）：V（3mol／L氨水）＝300：50：3：1为流动相,检测波长为280nm,流速为0．8mL／min。利培酮的线性范围为2～600μg／L（r＝0．996）,回收率为（98．2±3．5）％,日内与日间的标准偏差分别为4．12％和4．83％;9-羟利培酮的线性范围为2～800μg／L（r＝0．998）,回收率为（97．8±3．8）％,日内与日间的标准偏差分别为4．28％和4．81％。     

5-(对-羟基苯基):-5-苯基乙内酰脲的反相高效液相手性分离测定法

以β－ＣＤ为手性流动相添加剂、苯巴比妥为内标,于ＦＬＣ－Ｃ８反相柱上建立了鼠肝微粒体中５－（对－羟基苯基）－５－苯基乙内酰脲（ｐ－ＨＰＰＨ）外消旋体的拆分方法。测得ｐ－ＨＰＰＨ对映体的线性范围为０．５～１１０ｍｇ／Ｌ（ｒ＝０．９９９６）;最低检出量为５ｎｇ（Ｓ／Ｎ＝３）;Ｓ－ｐ－ＨＰＰＨ的回收率为９３．６％±２．８％,Ｒ－ｐ－ＨＰＰＨ的回收率为９４．７％±１．８％;日内和日间精密度ＲＳＤ值均小于２％。所建立的方法具有结果准确、操作方便等特点。     

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.     

Determination of metrifonate enantiomers in blood and brain samples using liquid chromatography on a chiral stationary phase coupled to tandem mass spectrometry

A novel enantioselective assay is described for the simultaneous determination of the metrifonate enantiomers BAY z 7216 and BAY z 7217 in extracts of whole blood samples obtained from rats, mice, rabbits and Beagle dogs as well as in rat brain tissue using liquid chromatography tandem mass spectrometry (LC/MS/MS) with thermally and pneumatically assisted electrospray ionization (TurboIonSpray(R)). Chromatographic separation is achieved on a chiral normal phase column with a mobile phase containing 0.25% water only. The total run time per sample is 11.0 min giving chromatographic base line separation of the enantiomers. Compared with previous methods this assay offers a higher sample throughput, excellent ruggedness and higher sensitivity. The limits of quantification for each enantiomer are 5.00 microg/L from 0.5 mL whole blood and 7.50 ng/g (ppb) using 0.333 g brain tissue, respectively. Similar assay specifications have been derived for the two enantiomers. The method has been validated for the analysis of blood samples from low and high dosed preclinical pharmacokinetic and toxicokinetic studies, corresponding to two analytical working ranges like e.g. 5.00 to 1000 microg/L and 200 to 40000 microg/L (0. 200 to 40.0 mg/L). For rat brain tissue the validated concentration range is 7.50 to 750 ng/g (ppb).     

离子色谱法分离分析Cr(Ⅲ)齐聚物

 用离子交换分析柱和二极管阵列检测器建立了水溶液中三价铬离子 [Cr(Ⅲ ) ]齐聚物的分析方法 ,研究了检测条件、流动相、离子强度对分析的影响 ,优化了分析条件。以 3mol/LNaClO4(内含 0 0 3mol/LHClO4)为流动相 ,在TSK GelSP 5PW离子交换分析柱 ( 75mm× 7 5mmi d ,10 μm)上对制备样品中的Cr(Ⅲ )齐聚物进行分离 ,以 2 0 0nm波长检测。该方法简便、快速 ,重复性好 ,10min内便可完成对制备样品中 3种Cr(Ⅲ )齐聚物的色谱分离。用该方法分析了各种制备条件下Cr(Ⅲ )齐聚物的含量 ,优化了制备条件。     

Simultaneous determination of palladium, platinum and rhodium by on-line column enrichment and HPLC with 5-(2,4-dihydroxyphenylazo)-rhodanine as pre-column derivatization reagent

In this paper, a new method for the simultaneous determination of palladium, platinum and rhodium ions was developed using a rapid column high performance liquid chromatography equipped with on-line enrichment technique. The palladium, platinum and rhodium ions were pre-column derivatized with DHAR to form colored chelates. The Pb-DHAR, Pt-DHAR and Rh-DHAR chelates could be absorbed onto the front of the enrichment column when they were injected into the injector and sent to the enrichment column [ZORBAX Stable Bound, 4.6 x 10 mm, 1.8 microm] with a 0.05 mol L(-1) of phosphoric acid solution as mobile phase. After enrichment, and by switching the six ports switching valve, the retained chelates were back-flushed by mobile phase and traveling towards the analytical column. The separation of these chelates on the analytical column [ZORBAX Stable Bound, 4.6 x 50 mm, 1.8 microm] was satisfactory with 54% acetonitrile (containing 0.05 mol L(-1) of phosphoric acid and 0.1% of tritonX-100) as mobile phase. Palladium, platinum and rhodium were separated completely within 2 min. By on-line enrichment technique, the enrichment factor of 100 was achieved, and the detection limits (S/N = 3) of palladium, platinum and rhodium reaches 1.4 ng L(-1), 1.6 ng L(-1) and 2.0 ng L(-1), respectively. This method was applied to the determination of palladium, platinum and rhodium in water, urine and soil samples with good results.     

反相高效液相色谱法测定人血浆中曲尼司特浓度

 报道了用反相高效液相色谱法测定人血浆中曲尼司特浓度。以甲醇－磷酸二氢钾缓冲液（６０∶４０，Ｖ／Ｖ；ｐＨ４．２）为流动相，色谱柱采用ＹＷＧ－Ｃ１８不锈钢柱，紫外３３３ｎｍ波长检测，血浆样品经甲醇沉淀蛋白后直接进样测定。血浆浓度在０．６５～４０ｍｇ／Ｌ范围内线性良好，方法的平均回收率为１００．０％±４．１％。血浆最低检测浓度为０．２ｍｇ／Ｌ。对１０名健康志愿者口服２００ｍｇ曲尼司特后不同时间的血药浓度进行了测定。     

Enantiomeric determination of L- and D-lactic acid in human cerebrospinal fluid by chiral ligand exchange high-performance liquid chromatography

Enantiomeric determination of L- and D-lactate in human cerebrospinal fluid (CSF) was achieved by HPLC on a chiral stationary phase with UV detection. Samples were submitted to a solid-phase extraction procedure using Oasis HLB Plus Extraction Cartridge and L- and D-lactate in the extract were separated by Shodex ORpac CRX-453 B column, a ligand exchange column for chiral separation, using a mobile phase containing copper (II) ion. L- and D-lactate were determined in 25 min. Intra-assay precision in CSF was 4.98% (mean 1.85 mmol/L) for L-lactate and 10.1% (mean 4.96 micromol/L) for D-lactate (n = 5). Detection limits were between 1.0 (L-lactate) and 1.5 (D-lactate) pmol. The mean values (n = 3) of analytical recovery for L- and D-lactate were 95% and 107%, respectively. The mean +/- SD of concentrations of L- and D-lactate in CSF (n = 20) were 1.52 +/- 0.54 mmol/L and 10.98 +/- 5.15 micromol/L, respectively.     

Direct determination of S-(-)- and R-(+)-propranolol glucuronide in rat hepatic microsomes by RP-HPLC

Propranolol, available commercially as a racemic mixture, is a non-selective beta-adrenergic blocking agent used in the treatment of hypertension, angina pectoris and cardiac arrhythmias. We have developed and validated an RP-HPLC assay method for direct determination of R-(+)- and S-(-)-propranolol glucuronide in rat hepatic microsomes to investigate the enantioselectivity of propranolol glucuronidation metabolism. A baseline separation of propranolol glucuronide enantiomers was achieved on a 5 microm reversed-phase ODS column, with a mixture of phosphate buffer (pH 3.5, 0.067 mol/L) and methanol (55:45, v/v) as mobile phase. Ultraviolet detection was set at 220 nm, and p-nitrobenzoic acid was used as internal standard. The standard curve of assay for R-(+)- and S-(-)-propranolol glucuronide in spiked microsomal incubate showed good linearity throughout the concentration range from 0.50 to 20.0 micromol/L. The analytical method affords average recovery of 99.8 and 100.1% for R-(+)- and S-(-)-propranolol glucuronide, respectively. The method provides a high sensitivity and good precision for R-(+)- and S-(-)-propranolol glucuronide (RSD < 10%). The LOD was 0.15 micromol/L and the LOQ was 0.5 micromol/L (RSD < 8%, n = 5) for both R-(+)- and S-(-)-propranolol glucuronide. The method is simple, precise and accurate, and is suitable for quantifying the propranolol glucuronides enantiomers in rat hepatic microsomes.     

Analysis of benzyldimethyldodecylammonium bromide in chemical disinfectants by liquid chromatography and capillary electrophoresis

Two novel analytical methodologies using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were developed and compared for the determination of benzyldimethyldodecylammonium bromide (BAB) in commercial compound chemical disinfectants. The LC analysis was performed with a Kromasil C18 (200 mm x 4.6 mm, 5 microm) column and a mobile phase of A:B = 80:20 (A: acetonitrile, B: 4 mmol/L octanesulfonic sodium--0.02 mol/L acetic sodium, adjusted with acetic acid to pH 5.2) at a flow rate of 1.0 mL/min. Detection was by ultraviolet absorption at 262 nm. The CE analysis was performed in a bare fused-silica capillary with 75 microm i.d. and total length of 46.4 cm with a buffer solution of 50% acetonitrile -50 mmol/L NaH2PO4, pH 2.24. The applied voltage was 20 kV. Detection was by ultraviolet absorption at 214 nm. Under optimized conditions, the HPLC retention time and CE migration time for BAB was 9.18 and 5.08 min, respectively. Calibration curves of peak area versus concentration gave correlation coefficients of 0.9996 for HPLC and 0.9994 for CE. The detection limits for HPLC and CE were 1.6 mg/L and 0.2 mg/L, respectively. Average recoveries at three concentration levels (50, 100, 200 mg/L for HPLC: 20, 40, 100 mg/L for CE) were 99.94 +/- 1.5, 99.64 +/- 1.3 and 99.61 +/- 0.4% for HPLC and 120.47 +/- 2.6, 102.06 +/- 8.7 and 103.05 +/- 3.0% for CE, respectively. Although both methods were shown to be suitable for the determination of BAB in commercial disinfectant compounds, CE provided analysis with less solvent purchase/disposal and better column efficiency, whereas HPLC provided superior precision.     

Hollow fiber-based liquid-phase microextraction (HF-LPME) of isradipine and its main metabolite followed by chiral HPLC analysis: application to an in vitro biotransformation study

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak® AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min^?1 was used. The column was kept at 23?±?2 °C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL^?1 and was linear over the concentration range of 50–5,000 and 50–2,500 ng mL^?1 for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.     

Determination of ofloxacin enantiomers in sewage using two-step solid-phase extraction and liquid chromatography with fluorescence detection

A robust analytical method has been developed and validated for the trace analysis of ofloxacin enantiomers in sewage using two-step solid-phase extraction purification and liquid chromatography with fluorescence detection (LC-FL). Ofloxacin enantiomers were separated on an Aglient TC-C-18 column using MeOH-water containing 4mmol/L CuSO4 and 5mmol/L l-isoleucine as mobile phase. The ofloxacin enantiomers were first extracted by a weak cation-exchange resin (WCX) and eluted with acidified MeOH (0.5% formic acid), then further purified by mixed mode of anion-exchange resin (MAX), resulting in ofloxacin recoveries generally above 95%. The limit of quantification was 0.08microg/L for each enantiomer. No significant matrix effect was found during the analytical procedure and standard solution calibration curves could be used for quantification. Total concentrations of both enantiomers in real sewage samples based on LC-FL method were consistent with those obtained upon liquid chromatography using tandem mass spectrometry (LC-MS/MS) method.     

Chiral determination of a novel acaricide cyflumetofen enantiomers in cucumber,tomato, and apple by HPLC

A simple chiral analytical method was developed for the enantiomeric determination of cyflumetofen in cucumber, tomato, and apple by normal‐phase HPLC. The effects of mobile phase composition and column temperature on the enantioseparation were evaluated. Excellent separation was achieved at 25°C on a Chiralpak AD‐H column, with a mixture of n‐hexane and 2‐propanol (95:5, v/v) as mobile phase at a flow rate of 1.0 mL/min detecting at 234 nm. The resolution of cyflumetofen enantiomers was up to 5.5. The elution order of the enantiomers was determined by an online OR‐2090 detector, which was performed under the same chromatographic conditions. The first eluted enantiomer was (–)‐cyflumetofen and the second eluted one was (+)‐cyflumetofen. The method was validated for linearity, repeatability, accuracy, LOD, and LOQ. LOD ranged from 0.1 to 0.15 mg/kg, with the LOD varying from 0.33 to 0.5 mg/kg for each enantiomer, respectively. The average recoveries of the pesticide ranged from 71.4 to 102.0% at all fortification levels. The precision values associated with the analytical method, expressed as RSD values, were below 14.8% in all matrices. The method was then successfully applied to detect cyflumetofen enantiomers in real samples.     

Simultaneous determination of four heavy metal ions in tobacco and tobacco additive by online enrichment followed by RP-HPLC and microwave digestion

A new method for the simultaneous determination of tin, lead, cadmium, and mercury in tobacco and tobacco additive by reversed-phase high-performance liquid chromatography combined with microwave digestion and an online enrichment technique is developed. The tin, lead, cadmium, and mercury ions are precolumn derivatized with tetra-(4-dimethylaminophenyl)-porphyrin (T(4)-DMAPP) to form color chelates. The Sn-T(4)-DMAPP, Hg-T(4)-DMAPP, Cd-T(4)-DMAPP, and Pb-T(4)-DMAPP chelates are absorbed onto the front of the enrichment column using a buffer solution of 0.05 mol/L pyrrolidine-acetic acid (pH = 10.0) as the mobile phase. After the concentration is finished (by switching the six-port switching valve) the retained chelates are back-flushed by the mobile phase and move to the analytical column. The chelate separation on the analytical column is satisfactory using gradient elution with methanol (containing 0.05 mol/L pyrrolidine-acetic acid buffer salt, pH = 10.0) and tetrahydrofuran (containing 0.05 mol/L pyrrolidine-acetic acid buffer salt, pH = 10.0). The linearity range is 0.01-120 micro g/L for each metal ion. The detection limits (S/N = 3) of tin, lead, cadmium, and mercury are 0.6, 0.8, 0.5, and 0.6 ng/L, respectively. This method is applied to the determination of tin, lead, cadmium, and mercury in tobacco and it's additive with good results.     

Determination of darusentan enantiomers in rat plasma by enantioselective liquid chromatography with tandem mass spectrometry using cellulose-based chiral stationary phase

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS/MS) method has been developed and validated for enantioselective determination of darusentan enantiomers, orally active potent endothelin-A receptor antagonist, in rat plasma. The plasma samples were pretreated by protein precipitation with methanol and baseline chromatographic separation was performed on a Chiralcel OD-RH column with a mobile phase consisting of acetonitrile/water/formic acid (50:50:0.1, v/v/v) at a flow rate of 0.5 mL/min. The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the negative ionization mode. The calibration curve was linear over the investigated concentration from 0.500 to 2500 ng/mL (r≥0.995) for each enantiomer using 50 μL of rat plasma. The lower limit of quantitation (LLOQ) for each enantiomer was 0.500 ng/mL. The intra- and inter-day precisions were not more than 10.2% and the accuracy was within the range from -5.4 to 6.3% for darusentan enantiomers. No chiral inversion was observed during the plasma preparation, storage and analysis. The method proved adequate for enantioselective pharmacokinetic studies of darusentan enantiomers after oral administration of three different doses of racemic darusentan.     

Capillary electrophoretic chiral determination of mirtazapine and its main metabolites in human urine after enzymatic hydrolysis

Capillary electrophoresis and liquid-phase microextraction using porous polypropylene hollow fibers were employed for the enantioselective analyses of mirtazapine and its metabolites demethylmirtazapine and 8-hydroxymirtazapine in human urine. Before the extraction, urine samples (1.0 mL) were submitted to enzymatic hydrolysis at 37 degrees C for 16 h. Then, the enzyme was precipitated with trichloroacetic acid, the pH was adjusted to 8 with 0.5 mol/L phosphate buffer solution (pH 11) and 15% sodium chloride was further added. The analytes were transferred from the aqueous donor phase, through n-hexyl ether (organic solvent immobilized in the fiber), into 0.01 moL/L acetic acid solution (acceptor phase). The electrophoretic analyses were carried out in 50 mmol/L phosphate buffer solution (pH 2.5) containing 0.55% w/v carboxymethyl-beta-cyclodextrin. The method was linear over the concentration range of 62.5-2500 ng/mL for each mirtazapine and 8-hydroxymirtazapine enantiomer and 62.5-1250 ng/mL for each demethylmirtazapine enantiomer. The quantification limit was 62.5 ng/mL for all the enantiomers. Within-day and between-day assay precision and accuracy were lower than 15% for all the enantiomers. Finally, the method proved to be suitable for pharmacokinetic studies.