A novel pre-column fluorescent derivatization method for the sensitive determination of aristolochic acids in medicinal herbs by high-performance liquid chromatography with fluorescence detection

Aristolochic acids (AAs) are a family of structurally related nitrophenanthrene carboxylic acids existing in Aristolochia, Bragantia, and Asarum species. AAs have been proven to have nephrotoxic and carcinogenic toxicity. In this study, a novel pre-column fluorescence derivatization procedure followed by high-performance liquid chromatography-fluorescence detection (HPLC-FLD) is developed for the analysis of AAs in medicinal herbs. The nitro group in the phenanthrene ring of AAs was removed by NaBH₄ in water–THF (2:1, v/v), resulting in the corresponding aristolic acids. The analysis of AAs in medicinal herbs was based of the sensitive fluorescence detection of aristolic acids after the chemical derivatization. Because the produced aristolic acids are highly fluorescent the limit of detection (LOD) of AAI and AAII were lowered to 0.06 and 0.04 ng/mL, respectively, which is at least an order of magnitude lower than those in the reported HPLC and LC–MS methods. Good linearity with correlation coefficients higher than 0.997 were obtained for AAI and AII in the calibration ranges of 0.2–800 ng/mL. The derivatization conditions such as reaction temperature, time and the amount of NaBH₄ were optimized. The developed method provided satisfactory intra-day and inter-day precisions with RSDs less than 1.4% and 3.8%, respectively. The relative analytical error was less than 7% for the analysis of AAI and AAII in spiked matrix samples.     

A fluorimetric liquid chromatographic method for the determination of propranolol in human serum/plasma

A simple, rapid, and sensitive fluorimetric-high-performance liquid chromatographic method for the determination of propranolol in human serum/plasma has been developed, without the need for solvent extraction. The procedure required 200 μl of serum/plasma, and the addition of 1 ml of acetonitrile for protein precipitation followed by vortexing and centrifugation at 10 000 g. The clear supernatant was evaporated to dryness under a stream of nitrogen at 50 60°C, the residue was reconstituted in 100 μl of methanol, and a 90 μl portion was injected onto the high-performance liquid chromatograph for propranolol quantitation. Chromatography was accomplished using a Hypersil^® cyano column, a mobile phase of acetonitrile - aqueous acetic acid (1%) containing 0.2% triethylamine (35:65, v/v) (pH 3.6), a flow rate of 1.5 ml min⁻¹, a fluorescence detector set at an excitation wavelength of 230 nm and an emission wavelength of 340 nm, and using pronethalol as the internal standard. Retention times for pronethalol and propranolol were 7.5 min and 9.5 min, respectively. Standard curves were linear in the range 5–200 ng ml⁻¹. Relative standard deviations for both inter-day and intra-day precision analysis were less than 7% for serum. No interference was observed from endogenous serum/plasma components. Specificity was shown for some, but not all, commonly coadministered drugs tested. The advantages of this method include good precision, low sample volume, good reproducibility and recovery, and high sensitivity.     

Fully automated methods for the determination of hydrochlorothiazide in human plasma and urine

LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology™ Robot and BenchMate™ Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid—liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate™ performed buffer and organic solvent addition, liquid—liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2–100 ng ml⁻¹ in plasma and 0. 1–20 μg ml⁻¹ in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.     

HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies

In this study, a simple, rapid and sensitive HPLC method with UV detection is described for determination of metformin in plasma samples from bioequivalence assays. Sample preparation was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed on a reversed-phase phenyl column at 40 degrees C. Mobile phase consisted of a mixture of phosphate buffer and acetonitrile at flow rate of 1.0 ml/min. Wavelength was set at 236 nm. The method was applied to a bioequivalence study of two drug products containing metformin, and allowed determination of metformin at low concentrations with a higher throughput than previously described methods.     

用HPLC法和微生物法测定人血浆中头孢丙烯浓度的比较

目的:比较用HPLC法和微生物法测定人血浆中头孢丙烯浓度的差异。方法:10名健康志愿者单剂量口服头孢丙烯片500 mg,分别用HPLC法和微生物法测定血浆中药物浓度。结果:HPLC法线性范围为0．143～14．300μg／mL,低、中、高浓度(0．357 5、1．430 0、7．150 0μg／mL)的回收率分别为(101．75±7．71)％、(96．89±2．55)％和(98．70±1．67)％(n= 6);3种浓度的日内、日间RSD分别为7．58％0、2．63％、1．69％和7．11％、3．77％、2．01％(n=6)。微生物法线性范围为0．25～3．00μg／mL,低、中、高浓度(0．50、1．75、3．00μg／mL)的回收率分别为(96．37±5．99)％、(108．26±8．39)％和(105．12±10．35)％(n=5);3种浓度的日内、日间RSD分别为9．84％、7．75％、6．22％和10．05％、8．31％、7．87％(n=5)。结论:两种方法回收率和精密度均符合要求,受试者各时间点平均血药浓度测定值无显著性差异。     

An improved HPLC method with fluorescence detection for the determination of pyrene in rat plasma and its pharmacokinetics

A high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) for the determination of pyrene in rat plasma was developed and validated. The method used fluorene as internal standard (IS), following a single-step protein precipitation, the analyte and internal standard were separated on a C18 column with a mobile phase containing methanol-water (90:10, v/v) at a flow rate of 1 ml/min. The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 265 and 394 nm, respectively. Two calibration curves were constructed in the range of 2-100 ng/ml and 0.1-5 microg/ml for pyrene with a lower limit of quantitation (LLOQ) of 2 ng/ml. Both intra-day and inter-day precision were less than 6% except at LLOQ, for which the precision was 10.6 and 9.8, respectively. Accuracy ranged from 98.3 to 103.6%, except at LLOQ, for which the accuracy was about 85%. The recovery ranged from 84.7 to 95.0% at the low, medium and high concentrations. The present HPLC-FLD method was rapid, sensitive, and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in female Wistar rats after administration of 10mg equivalent pyrene/kg dose of solution of pyrene and 1mg equivalent pyrene/kg dose of pyrene-loaded nanoparticle.     

高效液相色谱法测定万古霉素血药浓度

目的：建立人血浆中万古霉素ＨＰＬＣ测定法,为临床测血药浓度以调整万古霉素剂量提供方法学基础。方法：采用液－液萃取作提取法,并选用去甲万古霉素为内标,色谱条件：ＹＷＧ－ＲＰ１８分析柱,流动相为乙腈－０．０５ｍｏｌ／ＬＫＨ２ＰＯ４（８：９２）,紫外２３６ｎｍ检测。结果：本法在１．０５～８４μｇ／ｍｌ间线必耵关系数Ｒ＝０．０９９８（Ｎ＝５）,日内、日间精密度ＲＳＤ均〈１％。绝对回收率均〉７０％,结论：本     

HPLC-电化学法测定人血浆中盐酸克仑特罗浓度

目的　应用HPLC -电化学法检测人血浆中盐酸克仑特罗含量。方法　采用内标法定量,使用NucleodurC18色谱柱,以磷酸二氢钾缓冲液-乙腈(7∶3,V/V ,pH 5 .0 )为流动相,检测器电位0 .80V(Ag/AgCl)。结果　该方法的线性范围1.5～12 0μg·L-1,回归方程Y =0 .75 3X +0 .0 11,r=0 .9993;最低检测浓度0 .0 15 μg·L-1;RSD 2 .6 %～4 .2 % ;平均回收率93.4 %。结论　该方法适合临床监测     

High-performance liquid chromatographic assay for the derivatized enantiomers of propranolol and 4-hydroxypropranolol in human plasma

A stereospecific method for the analysis of propranolol and 4-hydroxypropranolol in human plasma employing fluorescence detection has been developed using the homochiral derivatizing agent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (TAGIT). The use of fluorescence detection provided enhanced sensitivity and cleaner chromatograms for the analysis of plasma samples, when compared to UV detection. Furthermore, parameters such as TAGIT concentration, vortex time and reaction time were examined to optimize conditions for maximum derivatization recoveries. The analyses of S(-)- and R(+)-propranolol from plasma were linear over the concentration range of 2.0-200 ng ml-1, while S(-)- and R(+)-4-hydroxypropranolol were linear from 5.0 to 200 ng ml-1.     

Fluorous derivatization and fluorous-phase separation for fluorometric determination of naproxen and felbinac in human plasma

Fluorous derivatization followed by fluorous-phase liquid chromatographic (LC) separation exploits the affinity between perfluoroalkyl compounds for highly selective and quantitative isolation of various analytes. However, the applicability of this technique as a simple pretreatment for fluorometric determination in clinical settings has not been fully explored. Here we show the applicability of this technique to the clinical determination of non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma. Naproxen and felbinac, widely used native-fluorescent NSAIDs with a carboxyl group, can have toxic effects at acute doses, and were therefore chosen as representative NSAIDs. Samples were precolumn derivatized with a non-fluorescent fluorous amine, which allowed highly selective retention of only derivatized substances in the fluorous LC column. Thus, subsequently, only the retained fluorous-labeled and fluorescent analytes were detected fluorometrically at appropriate retention times. The detection limits for these two drugs were less than 11fmol on column. Correlation curves were liner over the range of 0.04-10 and 5-250nmol/mL plasma for both two drugs (r>0.999) with good repeatability. Thus, this method offers a simple, sensitive, and selective solution for determination of NSAIDs in clinical settings.     

一种改进的HPLC法测定人血浆中盐酸二甲双胍浓度

目的采用改进的离子对高效液相色谱方法测定人血浆中二甲双胍的浓度.方法乙腈作为蛋白沉淀剂,除去酸化血浆样品中的蛋白,二氯甲烷进一步去杂质后,取上清液进样.流动相为甲醇(56%),0.35 mmol·L-1的十二烷基硫酸钠和5mmol·L-1的磷酸盐缓冲液(44%,pH 6.6),流速为1.2mL·min-1,紫外检测波长234 nm.结果二甲双胍和内标(哌仑西平)的保留时间分别为7.1和4.8 min,标准曲线在20～4 000mg·L-1浓度范围内线性关系良好,绝对回收率＞95%,检测限和定量限分别为4和10 mg·L-1.天内和天间RSD＜6.4%.结论该方法适用于盐酸二甲双胍的治疗药物监测及临床药学研究.     

A fully automated high-performance liquid chromatographic method for the determination of indomethacin in plasma

A fully automated method is described, which enables the determination of indomethacin in plasma by reversed-phase HPLC following on-line sample enrichment and clean-up on a short pre-column.

The plasma sample is introduced directly into the column switching system. The pre-column, filled with a pellicular bonded phase, is first washed with phosphate buffer, pH 7.4. The compounds retained on the pre-column are then eluted in the fore-flush mode and separated on an octadecylsilica column with a methanolic phosphate buffer (pH 7.4) mobile phase. Indomethacin is determined spectrophotometrically at 254 or 260 nm.

The effect of changes in the pH and flow rate of the washing eluent are studied. Under the conditions selected, memory effects can be avoided, the absolute recovery of the drug is 70% and the limit of detection 10 ng ml⁻¹ for a 100 μl injection of plasma. At a concentration of 100 ng ml⁻¹, the relative standard deviations (RSD) are 1.7% (within-day) and 3.5% (between-day), respectively.     

LC-MS/MS法同时测定CUMS大鼠血浆中3种单胺类神经递质

目的建立测定大鼠血浆中五羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)的LC-MS/MS方法,观察慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)抑郁大鼠血浆单胺类神经递质DA、5-HT、NE的变化情况。方法♂SD大鼠22只,分为对照组(n=10)和模型组(n=12),模型组每天给予9种慢性不可预见性温和刺激因子,造模21 d后,分别于造模前后进行行为学测定及眼眶采血;采用苯甲酰氯作为柱前衍生化试剂,将3种待测物及内标衍生化后进入LC-MS/MS检测,测定造模前后血浆中5-HT、NE、DA 3种神经递质浓度。结果造模21d后,与对照组相比,模型组大鼠体重明显降低(P<0.05),水平得分、垂直得分均明显降低(P<0.01),同时糖水消耗量明显降低(P<0.01)。血浆中5-HT、NE、DA的浓度在1.47~752、1.75~898、2.05~1053μg·L-1范围内线性关系良好,最低定量限分别为1.47、1.75、2.05μg·L-1,以质控样品计算,在各浓度水平下,此法的回收率均大于70%,日内和日间精密度小于15%,符合生物样品分析要求。造模21 d后,模型组血浆中5-HT、NE、DA的浓度分别为(3.99±1.21)、(6.24±1.94)、(6.07±1.98)μg·L-1,与对照组相比均明显降低(P<0.01)。结论 CUMS造模成功后,血浆中的3种神经递质分别呈下降趋势。     

Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma

A simple, sensitive and rapid chromatographic method was developed and validated for the simultaneous quantification of atenolol and chlorthalidone in human plasma using hydrochlorothiazide as internal standard (IS). The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyl column with isocratic elution with 10 mM KH₂PO₄ (pH 6.0) – methanol (70:30, v/v) at ambient temperature with flow rate of 1 mL min⁻¹ and UV detection at 225 nm. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range of 0.1–10 μg mL⁻¹. The method was validated in terms of accuracy, precision, absolute recovery, freeze–thaw stability, bench-top stability and re-injection reproducibility. The within- and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze–thaw cycles (deviation <15%). The proposed method was specific for the simultaneous determination of atenolol and chlorthalidone in human plasma where there was no interference from endogenous biological substances.     

Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.     

HPLC法检测肾移植病人全血中西罗莫司的药物浓度

目的 :建立测定全血中西罗莫司含量的方法。 方法 :采用HPLC法 ,色谱柱为 :YMC PackODS A(15 0mm× 4 .6mm ,5 μm) ,预柱为AlltimaC18(7.5mm× 4 .6mm ,5 μm)。流动相为乙腈∶四氢呋喃∶水 =5 5∶5∶4 0 ;检测波长为 2 78nm ,流速为 1.5ml/min ,柱温 :5 0℃。 32 去甲氧基西罗莫司为内标。 结果 :西罗莫司及内标 32 去甲氧基西罗莫司的保留时间分别为 10 .1、12 .1min ,全血定量线性范围 :2 .4 9～ 76 .36ng/ml,最低检测浓度为 1.98ng/ml,方法回收率为 99.93%～ 10 5 .90 % ,日内、日间RSD <7.4 0 %。 结论 :本方法准确、可靠 ,适用于临床对西罗莫司的血药浓度监测。     

用RP-HPLC法同时测定拉莫三嗪和奥卡西平的血药浓度

目的：用RP-HPLC法同时测定人血浆中抗癫药拉莫三嗪和奥卡西平的浓度。方法：以艾司唑仑为内标,色谱柱为Agilent Zorbax XDB-C18柱（150 mm×4.6 mm,5μm）;流动相为甲醇-0.1%三氟乙酸（38∶62）;流速为1.0 mL/min;检测波长为240 nm。结果：拉莫三嗪和奥卡西平的血药浓度分别在1.00-50.00μg/mL（Y=0.214 6c-0.010 9,r=0.999 4）和0.50-50.00μg/mL（Y=0.396 6c＋0.006 1,r=0.999 1）范围内线性良好;平均回收率分别为97.64%和97.67%（n=5）。拉莫三嗪2.5、10.0和40.0μg/mL的日内RSD分别为4.6%、3.7%和2.8%（n=5）,日间RSD分别为5.9%、6.4%和4.8%（n=5）;奥卡西平1.0、10.0和40.0μg/mL的日内RSD分别为3.6%、3.4%和4.0%（n=5）,日间RSD分别为5.5%、3.7%和4.3%（n=5）。结论：本方法操作简单,回收率高,精密度好,适用于临床上常规监测拉莫三嗪和奥卡西平的血药浓度。     

一种简易测定人血浆氧氟沙星浓度的高效液相色谱法

目的　建立一种简易的测定人血浆氧氟沙星浓度高效液相色谱法。方法　血浆由蛋白沉淀剂[甲醇∶ 5 % Zn SO4 (70∶ 30 ,v/v) ]沉淀蛋白后上清液直接进样。所用色谱柱为 Hypersil BDS C1 8柱 ,流动相为0 .0 5 mol/L 柠檬酸缓冲液 (三乙胺调 p H值到 4 .0 )∶乙腈 (83∶ 17,v/v) ,荧光检测器激发波长为 2 95 nm,发射波长为 5 0 5 nm。结果　直接沉淀蛋白后样品干净 ,色谱分离效果好 ,无内源性杂质干扰。在 0 .0 1～ 15 μg/ml浓度范围内线性关系好。提取回收率为 76 .3% ,日内精密度和日间精密度均小于 10 %。结论　本法简便、可靠 ,可用于血药浓度测定。