A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

Simultaneous determination of glucose, 1,5-anhydro-d-glucitol and related sugar alcohols in serum by high-performance liquid chromatography with benzoic acid derivatization

A new, simple and sensitive pre-column high-performance chromatographic method for the determination of diabetes marker d-glucose, 1,5-anhydro-d-glucitol and related compounds is reported. Sugars (d-glucose, d-galactose, d-mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80 degrees C for 60 min. l-Fucose, fructose, d-lactose, l-rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo-inositol) were also derivatized with BA at 80 degrees C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. x 250 mm, 5 microm) with acetonitrile-50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was lambda(ex) 275 nm and lambda(em) 315 nm. The detection limits of sugars were 10-80 microg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 microg/mL sugars were 7.0-7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B-Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in microg/mL obtained by the proposed pre-column HPLC and enzyme-photometric method, respectively. The detection limits of sugar alcohols were 100-1000 ng/mL. The calibration graphs were linear to 50 microg/mL and relative standard deviations of 10 microg/mL were 7.2-8.2%. The 1,5-AG data by the proposed method was also compared with the enzymatic photometric 1,5-AG analysis method (Rana AG 1,5-AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d-glucose, 1,5-AG and related sugar alcohols in serum from healthy males.     

Sensitive determination of MDMA and its metabolite MDA in rat blood and brain microdialysates by HPLC with fluorescence detection

Simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in rat blood and brain microdialysates by high-performance liquid chromatography with fluorescence detection (HPLC-FL) was developed. Microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The DIB-derivatives of MDMA, MDA and the internal standard, 1-methyl-3-phenylpropylamine (MPPA), were isocratically separated on an ODS column using a mixture of 50 mm phosphate buffer (pH 7.0)-acetonitrile-methanol-2-propanol (50:45:5:2, v/v/v/v %) as an eluent at a flow rate of 1.5 mL/min. The calibration curves of MDA and MDMA spiked to blood and brain microdialysates were linear over the ranges 2.5-500 and 5.0-1000 ng/mL, respectively. The detection limits of MDA and MDMA were 1.2 and 4.2 for blood and 1.3 and 4.8 ng/mL for brain, respectively. Additionally, the intra- and the inter-assay precisions were lower than 5.6% for the blood and brain microdialysates (n = 4). The proposed method was successfully applied for the monitoring of MDMA and its metabolite MDA in rat blood and brain microdialysates, and the pharmacokinetic parameters of MDMA and MDA in the microdialysates after administration of MDMA (5 mg/kg, i.p.) with or without caffeine (20 mg/kg, i.p.) were evaluated.     

A new HPLC method with fluorescence detection for the determination of memantine in human plasma

A sensitive, selective, simple and fast HPLC method based on the formation of derivative with fluorescamine was developed for the determination of memantine (ME) in human plasma. Separation was achieved on a CN column (200 mm×4.6 mm) using acetonitrile-10 mM orthophosphoric acid containing 1 mL/L triethylamine (45:55, v/v) at a flow rate of 1 mL/min. Emission and excitation wavelengths were 480 and 380 nm, respectively. Amantadine was used as an internal standard. Calibration graphs were rectilinear over the range of 1.0-100.0 ng/mL. Limit of detection and limit of quantification were found to be 0.3 and 1.0 ng/mL, respectively. Intra-day and inter-day relative standard deviation values were found to be <2.03%. Average recovery was also found to be around 94%. Proposed method was applied for the pharmacokinetic study in a healthy volunteer after a single oral administration of 20 mg of ME.     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

Monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.     

铽增敏高效液相色谱柱后衍生荧光检测法同时检测鸡肉中的三种氟喹诺酮类药物残留

基于氟喹诺酮类药物与铽离子形成配合物后的荧光增强作用,建立了同时检测鸡肉中氟喹诺酮类(FQs)药物环丙沙星、诺氟沙星和恩诺沙星残留的Tb3+增敏高效液相色谱（HPLC）柱后衍生荧光检测方法。优化的实验条件如下：流动相为0.05 mol/L 醋酸/醋酸钠缓冲液(pH 6.0)-乙腈(体积比为89∶11),色谱柱为Hypersil BDS-C18,柱温40 ℃,流速0.8 mL/min;Tb3+浓度为8×10-5 mol/L;衍生反应温度40 ℃,衍生泵流速0.5 mL/min;荧光检测激发波长271 nm,发射波长545 nm。实验结果表明,将上述3种药物以1.0,10.0,50.0,100.0 ng/g水平添加到鸡肉后的回收率范围为66.3%~88.0%,相对标准偏差（RSD）均小于15.0%。定量分析的线性范围为0.1~500 ng/mL,方法的日内和日间RSD均小于13.0%;最低检出限分别为0.05(环丙沙星)、0.05(诺氟沙星)和0.08(恩诺沙星)ng/g,比前人报道的非衍生高效液相色谱荧光检测法检测FQs药物的灵敏度有极大的提高。该项研究为FQs药物多残留检测提供了灵敏度更高的新方法。     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

Nonextractive procedure and precolumn derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for trace determination of trimetazidine in plasma by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed and validated in a single laboratory for the trace determination of trimetazidine (TMZ) in human plasma. Fluoxetine (FLX) was used as the internal standard. TMZ and FLX were isolated from plasma by protein precipitation with acetonitrile and derivatized by heating with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole in pH 8 borate buffer at 70 degrees C for 30 min. Separations were performed in the isocratic mode on a Nucleosil CN column with the mobile phase acetonitrile-10 mM sodium acetate buffer (pH 3.5)-methanol (47 + 47 + 6, v/v/v) at a flow rate of 1.0 mL/min. The derivatized samples were excited at 470 nm and monitored at an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with a good correlation coefficient (r = 0.9997, n = 5) was obtained for the peak area ratio of TMZ to FLX and for TMZ concentrations of 1-120 ng/mL. The proposed method has the lowest limits of detection and quantitation reported to date for the determination of TMZ in plasma with values of 0.3 and 0.95 ng/mL, respectively. The values for intra- and interassay precision were satisfactory; the relative standard deviations were < or =4.04%. The accuracy of the method was demonstrated; the recoveries of TMZ from spiked human plasma were 98.13-102.83 +/- 0.2-4.04%. The method has high throughput because of its simple sample preparation procedure and short run time (<10 min). The results demonstrated that the proposed method would have great value when applied in pharmacokinetic studies for TMZ.     

A validated sensitive HPLC‐MS/MS method for quantification of a potential hypnotic drug MT502 and its application to a pharmacokinetic study in rat

A rapid, sensitive HPLC‐MS/MS method was established and validated to assay the concentration and pharmacokinetic profile of MT502, a promising hypnotic drug. The plasma sample was treated by a liquid–liquid extraction and separated on a kromasil C₁₈ column at an isocratic flow rate of 0.3 mL/min using methanol and 0.1% formic acid in water (75:25, v/v) as mobile phase. The mass spectrometric detection was carried out using a triple‐quadrupole system via positive electrospray ionization. Multiple reaction monitoring was used for quantitation of m/z transitions from 261 to 188 for MT502 and from 247 to 188 for MT501 (internal standard). Good linearity was achieved over the concentration range of 1–1000 ng/mL and 10–5000 ng/mL with lower limit of quantification of 0.30 and 0.80 ng/mL. The intra‐ and inter‐day precisions, accuracy, recovery and stability were satisfactory for the concentration test. The above method can be used for a pharmacokinetic study at doses of 1, 5 and 20 mg/kg. Results indicated that MT502 had rapid absorption, rapid elimination and linear pharmacokinetic properties within the range of the tested intragastric dose. This developed HPLC‐MS/MS method was successfully applied to a pharmacokinetic study of MT502 for the first time and was demonstrated to be simple and sensitive. Copyright © 2015 John Wiley & Sons, Ltd.     

Validation and application of a high-performance liquid chromatography/tandem mass spectrometry assay for sumatriptan in human plasma

A sensitive and convenient high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) assay is described for the (5-HT(lB/lD)) receptor agonist sumatriptan in human plasma. Sumatriptan was recovered from plasma (81.8 +/- 6.8%) by liquid-liquid extraction. The mobile phase flow rate was 0.3 mL/min and consisted of methanol:water:formic acid (90:10:0.1, v/v/v). The analytical column (4.6 x 100 mm) was packed with Partisil C(8) (5 micro m). The standard curve was linear from 0.7 to 70.4 ng/mL (r(2) > 0.99). The lower limit of quantitation was 0.7 ng/mL. The assay was specific, accurate (percentage deviation from nominal concentrations were <15%), precise and reproducible (within- and between-day coefficients of variation <10.3%). Sumatriptan in plasma was stable over three freeze/thaw cycles and at room temperature for one day. The utility of the assay was demonstrated by following sumatriptan plasma concentrations in two healthy subjects for 8-12 h following a single 20 mg intranasal dose.     

Determination and validation of duloxetine hydrochloride in capsules by HPLC with pre-column derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of duloxetine hydrochloride in capsules. The method was based on pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole using the fluorimetric detection technique. Duloxetine hydrochloride was analyzed by HPLC using an Inertsil C18 column (5 μm, 150 × 4.6 mm) and mobile phase consisted of methanol and water (65:35, v/v). The fluorescence detector was adjusted at excitation and emission wavelengths of 461 and 521 nm, respectively. The linearity of the method was in the range of 10-600 ng/mL. Limits of detection and quantification were 0.51 and 1.53 ng/mL, respectively. The proposed method was successfully applied for determination of duloxetine hydrochloride in its pharmaceutical preparation. The results were in good agreement with those obtained using a reference method.     

Determination of faropenem in human plasma and urine by liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS.     

High-performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization

In this study, we investigated a simple, sensitive and reliable liquid chromatography‐fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl). For the first time, FMOC‐Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC‐Cl to form memantine hydrochloride‐FMOC‐Cl and amantadine hydrochloride‐FMOC‐Cl compositions, which can be very compatible for LC‐FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C₁₈ column (DIAMONSIL 150×4.6 mm, id 5 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride‐FMOC‐Cl and amantadine hydrochloride‐FMOC‐Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025–5.0 μg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra‐day and inter‐day assays were 4.46–12.19 and 5.23–11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.     

Precolumn fluorescence labeling method for simultaneous determination of hydroxyzine and cetirizine in human serum

A highly selective and sensitive method was developed for simultaneous determination of the antihistaminic drug hydroxyzine (HZ) and its pharmacologically active metabolite cetirizine (CZ) in human serum using haloperidol as internal standard. The method was based on fluorescence labeling of both drugs with a fluorescent arylboronic acid 4-(4,5-diphenyl-1H-imidazol-2-yl)phenyl boronic acid followed by separation on silica column using a mobile phase consisting of acetonitrile and water (90:10, v/v%) containing triethylamine and acetic acid. The labeling reaction conditions were optimized and the liquid-liquid extraction method was successfully applied to extract the both drugs from serum. The linearity range was 0.025-2.00 microg/mL for HZ and CZ. The limit of detection (S/N = 3) was 10 and 5 ng/mL for HZ and CZ, respectively.     

超高效液相色谱法同时测定纺织品中18种荧光增白剂

建立了同时测定纺织品中18种荧光增白剂（FWAs）的超高效液相色谱-荧光检测（UPLC-FLR）法。试样由三氯甲烷-乙醇（6：4,v/v）超声提取,以ACQUITY UPLC HSS T3柱（100 mm×2.1 mm,1.8 μm）为分离色谱柱进行分析,以5 mmol/L乙酸铵水溶液和甲醇为流动相,梯度洗脱,流速为0.4 mL/min,荧光激发波长为350 nm,发射波长为430 nm,外标法定量。18种FWAs在各自范围内呈良好的线性关系,相关系数（R²）均≥ 0.9992;方法的定量限（LOQs,S/N=10）为0.002~0.1 mg/L。样品的平均加标回收率为88.3%~104.5%,相对标准偏差（RSD）为2.0%~5.5%（n=6）。该方法灵敏度高,精密度好,准确度高,适用于各种纺织品中FWAs的测定。     

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

Determination of sertraline in rat plasma by HPLC and fluorescence detection and its application to in vivo pharmacokinetic studies

A simple, rapid, and sensitive HPLC method based on 9H‐fluoren‐9‐ylmethyl chloroformate derivatization for the quantification of sertraline in rat plasma has been developed, requiring a plasma sample of only 0.1 mL, which was deproteinized and derivatized for 5 min in two single steps. The obtained derivative was stable at room temperature and was determined by HPLC using a fluorescence detector. The analytical column was a C(18) column and the mobile phase was acetonitrile and water (80:20, v/v). Calibration curves were linear in the range of 10–500 ng/mL. The limit of detection was approximately 3 ng/mL, and the lower limit of quantification was established at 10 ng/mL. The bias of the method was lower than 10%, and the within day as well as between day, relative standard deviations were lower than 12%. This analytical method was successfully applied to characterize sertraline pharmacokinetics in rats following intravenous (t_1/2 = 213 ± 48 min, Cl = 43.1 ± 8.7 mL/min, V_d = 11560 ± 1861 mL) and oral (C_max = 156 ± 76 ng/mL, t_max = 63.8 ± 16.3 min) administration of 2 and 5 mg, respectively.     

Determination of piceatannol in rat serum and liver microsomes: pharmacokinetics and phase I and II biotransformation

A method of analysis of piceatannol in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of piceatannol and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL), were precipitated with acetonitrile after addition of the internal standard, 4-methylumbelliferone. Separation was achieved on a phenomenex C(18) column (250 x 4.6 mm i.d., 5 microm) equipped with a phenomenex C(18) (4 x 3.0 mm i.d., 5 microm) guardcolumn with fluorescence excitation at 320 nm and emission at 420 nm. Separation was also possible with UV detection at 310 nm. The fluorescent calibration curves were linear ranging from 0.05 to 100 microg/mL. The mean extraction efficiency was >95%. Precision of the assay was <10% (coefficient of variation), and was within 10% at the limit of quantitation (0.05 ng/mL). Bias of the assay was lower than 7%. The limit of detection was 50 ng/mL for a 0.1 mL sample. The assay was applied successfully to the in vitro kinetic study of metabolism of piceatannol in rat liver microsomes and pharmacokinetics in rats. Three metabolites of piceatannol have been identified. .     

Determination of paeoniflorin in rat plasma by a liquid chromatography-tandem mass spectrometry method coupled with solid-phase extraction

A rapid, sensitive and selective liquid chromatography-tandem spectrometry method was developed and validated for determination of paeoniflorin in rat plasma using geniposide as the internal standard. The samples were pretreated with solid-phase extraction using Extract-Clean cartridges. Separation of paeoniflorin and IS was achieved on a reversed-phase C18 column (50x4.6 mm i.d.) with a mobile phase made up of acetonitrile and 0.05% formic acid (25:75, v/v) at a flow rate of 0.5 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring and an electrospray ionization source was employed as the ionization source. The lower limit of quantification obtained was 4 ng/mL (n=6) using 200 microL plasma with an accuracy of -3.67% (relative error) and a precision of 4.13% (relative standard deviation). A good linearity was found in the range of 4-1000 ng/mL. The intra- and inter-day relative standard deviations in the measurement of quality control samples 10, 150 and 800 ng/mL ranged from 3.73 to 4.94% and from 4.31 to 6.56%, respectively. The accuracy was from -3.93 to -1.11% in terms of relative error. The analyte and IS were stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of paeoniflorin after a single oral administration of 53.36 mg/kg paeoniflorin to rats.