Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

A reversed-phase high-performance liquid chromatographic method for the determination of zafirlukast in pharmaceutical formulations and human plasma

Zafirlukast (ZAF) is a leukotriene receptor antagonist used in the treatment of chronic asthma. In this study, a simple and sensitive reversed-phase, high-performance liquid chromatographic method was developed for the determination of ZAF in pharmaceutical formulations and human plasma. Piribedil was used as an internal standard. Analysis was carried out on a Nucleosil C18 100 A (150 mm x 4.6 mm id, 5 Vm) column with acetonitrile-pH 3.0 acetate buffer (70 + 30, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The peak was detected by an ultraviolet detector set at a wavelength of 240 nm. The retention times were about 3.9 min for piribedil and 5.8 min for ZAF. The developed method was applied to the determination of ZAF in its pharmaceutical formulation and spiked human plasma. For quantification of ZAF in spiked plasma, proteins were precipitated with ethanol before chromatographic analysis. The calibration range was linear from 49.69-437.50 ng/mL in spiked plasma. The absolute recovery from spiked plasma was 98.73 +/- 0.42% at a concentration of 254.78 ng/mL of ZAF. No endogenous substances from plasma were found to interfere.     

Enantiomeric determination of praziquantel and its main metabolite trans-4-hydroxypraziquantel in human plasma by cyclodextrin-modified micellar electrokinetic chromatography

A capillary electrophoresis method for the simultaneous quantitation of praziquantel and its main metabolite trans-4-hydroxypraziquantel enantiomers in human plasma was developed and validated using cyclodextrin-modified micellar electrokinetic chromatography. Sample clean-up involved a single-step liquid-liquid extraction of plasma with toluene after the addition of NaCl. The complete enantioselective analysis was obtained in less than 7 min using 2% w/v sulfated beta-cyclodextrin as chiral selector and 20 mmol/L sodium deoxycholate as surfactant, in 20 mmol/L sodium borate buffer, pH 10. A 50 microm x 42 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 18 kV and at 20 degrees C. The calibration curves were linear over the 125-625 ng/mL concentration range. The mean recoveries for praziquantel and trans-4-hydroxypraziquantel were up to 96 and 71%, respectively, with good precision. All four enantiomers were quantified at two concentration levels (200 and 600 ng/mL) with precision and accuracy below 15%. The quantitation limit was 50 ng/mL for (-)-(R)- and (+)-(S)-praziquantel and 62.5 ng/mL for (-)-(R)- and (+)-(S)-trans-4-hydroxypraziquantel, using 1 mL of human plasma.     

高效液相色谱-荧光检测法测定罂粟籽和火锅汤料中的罂粟碱

用所建立的高效液相色谱-荧光检测法测定了罂粟籽和火锅汤料中的罂粟碱。采用的色谱柱为RP-C18柱(250 mm×4.6 mm i.d.,5 μm);检测激发波长为285 nm,发射波长为355 nm;流动相为甲醇-0.02 mol/L乙酸铵(体积比为70∶30),流速0.8 mL/min。实验结果表明,罂粟碱的进样量为1×10-4~0.1 μg时其质量浓度与相应峰面积有良好的线性关系,最低检测限(以信噪比大于3计)达到0.02 ng。罂粟籽中罂粟碱的回收率为99.0%～100.8%。方法快速准确,简便灵敏,分离度高,能够满足有关食品中罂粟碱的检测要求。     

Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.     

Selective liquid chromatographic method for determination of fluoxetine in plasma.

A selective and sensitive liquid chromatographic method was developed for the determination of fluoxetine (FLU) in plasma. FLU was isolated from plasma by liquid-liquid extraction. The chromatographic separation was performed on an analytical 250 x 3.9 mm id Novapak C18 column (4 microm particle size) with an isocratic mobile phase consisting of phosphate buffer-acetonitrile-methanol -triethylamine (58 + 30 + 10 + 2, v/v) adjusted to pH 7. Using UV detection at 226 nm, the detection limit for FLU in plasma was 3 ng/mL. No interferences were found with tricyclic antidepressant drugs, which allows this method to be used in clinical studies. The calibration curve was linear over the concentration range of 10-200 ng/mL. The average recovery was about 80% for plasma. The inter- and intraday assay coefficients of variation were <8%.     

Simultaneous quantitative analysis of ketanserin and ketanserinol in plasma by RP-HPLC with fluorescence detection

A sensitive and selective high-performance liquid chromatographic assay for the quantification of ketanserin and ketanserinol in human plasma was developed and validated. The procedure involves extraction of ketanserin and ketanserinol from plasma using an Extrelut NT-1 solid-phase extraction column. The chromatograph was equipped with a Hypersil BDS column (100 x 4.5 mm, 3 micro m particle size). Separation was performed with a mixture of acetate buffer 0.01 M, pH 4.9-methanol-acetonitrile (52:40:8, v/v/v). Detection was performed with fluorescence detection (lambda(ex) = 332 nm and lambda(em) = 410 nm). Calibration curves were linear (r(2) = 0.999) in the range 0-400 ng/mL for both ketanserin and ketanserinol. The repeatability coefficient for ketanserin and ketanserinol was 3.1 and 3.0%, respectively. The reproducibility coefficient for ketanserin and ketanserinol was 10.5 and 9.1%, respectively. The limit of quantification for both ketanserin and ketanserinol was 2.0 ng/mL. The mean recovery yield for both ketanserin and ketanserinol was 60%. In an 8 h work day approximately 60 samples, including calibration and reference standards, could be processed.     

High-performance liquid chromatography analysis of curcumin in rat plasma: application to pharmacokinetics of polymeric micellar formulation of curcumin

A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of curcumin in rat plasma. Plasma was precipitated with acetonitrile after addition of the internal standard (IS), 4-hydroxybenzophenone. Separation was achieved on a Waters muBondapak C(18) column (3.9 x 300 mm, 5 microm) using acetonitrile (55%) and citric buffer, pH 3.0 (45%) as the mobile phase (flow rate = 1.0 mL/min). The UV detection wavelength was 300 and 428 nm for IS and curcumin, respectively. The extraction efficiencies were 97.08, 95.69 and 94.90% for 50, 200 and 1000 ng/mL of curcumin in rat plasma, respectively. The calibration curve was linear over the range 0.02-1 microg/mL with a correlation coefficient of r(2) > 0.999. The intra- and inter-day coefficients of variation were less than 13%, and mean intra- and inter-day errors were less than +/-6% at 50, 200 and 1000 ng/mL of curcumin. This assay was successfully applied to the pharmacokinetic studies of both solubilized curcumin and its polymeric micellar formulation in rats. It was found that polymeric micelles increased the half-life of curcumin 162-fold that of solubilized curcumin and increased the volume of distribution (Vd(ss)) by 70-fold.     

Direct injection determination of benzoylecgonine, heroin, 6-monoacetylmorphine and morphine in serum by MLC

A simple and sensitive direct injection chromatographic procedure is developed for the determination of heroin, two of its metabolites (morphine and 6-monoacetylmorphine (6-MAM)), and benzoylecgonine (a metabolite of cocaine) in serum samples. The proper resolution of the four substances is obtained with a chemometrics approach, where the retention is modelled as a first step using the retention factors obtained in a limited number of mobile phases. Afterwards, an optimisation criterion that takes into account the position and shape of the chromatographic peaks is applied. The mobile phase selected to carry out the analysis was 0.1 mol L(-1) SDS-4% (v/v) butanol buffered at pH 7, in which the separation is performed in less than 18 min. The limits of quantification were in the 17-36 ng mL(-1) range. Intra- and inter-day assay accuracy and precision (below 3%) were obtained following ICH guidelines. The method developed was applied to the determination of the drugs studied in serum samples with good recoveries (90-104%). Serum samples from subjects that have been ingested cocaine and heroin were also analysed. The samples were injected directly in the chromatographic system without any pretreatment.     

液相色谱-串联质谱法测定人血浆中阿扑吗啡

盐酸阿扑吗啡(apomorphinehydrochloride)为吗啡的衍生物,系中枢多巴胺D2受体激动剂,其舌下片制剂是目前用于治疗男性勃起机能障碍的新药。为了研究该制剂中阿扑吗啡的药代动力学特性,我们建立了阿扑吗啡血药浓度测定的液相色谱-串联质谱法。测定了28名健康志愿者口服阿扑吗啡舌下片后阿扑吗啡的血药浓度。已有文献报道采用HPIC-UV、HPLC-荧光、HPLC-电化学和GC等方法测定血浆中阿扑吗啡浓度,在灵敏度方面均低于我们所建立的方法。     

Capillary zone electrophoresis (CZE) coupled to time-of-flight mass spectrometry (TOF-MS) applied to the analysis of illicit and controlled drugs in blood

A new method for the determination of illicit and abused drugs in blood by capillary zone electrophoresis-electrospray ionization-time-of-flight mass spectrometry is proposed, in view of its application in clinical and forensic toxicology. The analytes (methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine, 6-acethylmorphine, benzoylecgonine) were separated with capillary zone electrophoresis by applying 15 kV within 25 min, in an uncoated fused-silica capillary (75 microm x 100 cm) using a 25 mM ammonium formate electrolyte solution (pH 9.5). The capillary electropherograph was coupled to time-of-flight mass spectrometry through an orthogonal electrospray ionization source, with a coaxial sheath liquid interface. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 0.5% formic acid delivered at 4 microL/min. Forensic drugs were identified by exact mass determination (mass accuracy typically < or =5 ppm) and by matching of the isotopic pattern. Under optimized conditions, linearity was assessed in the range 10-2000 ng/mL, with correlation coefficients between 0.9744 and 0.9982 for all the analytes. LODs were in the range of 2-10 ng/mL (S/N > or =3) and LOQs of 10-30 ng/mL. The CVs (tested at 40 and 800 ng/mL in biological matrix) were below 2.97% for migration times and below 14.61% for peak area ratios (analyte/internal standard). Blood samples were extracted by using a liquid-liquid extraction procedure and injected under field-amplified sample stacking conditions. The method was successfully applied to real cases.     

Determination of triptolide and triptonide in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method for determination of triptolide and triptonide in human plasma is described. Plasma samples were extracted with OasisHLB solid-phase extraction (SPE) cartridges. After pretreatment, they were separated on a SymmetryShieldRP(18) column with a mobile phase of acetonitrile-water (40:60,v/v) at 40 degrees C. The effluent was monitored at UV 217 nm. Linearity (0.010-1.0 mg/L) was good, and the lower limit of detection was 3 ng/mL for triptolide and 4.5 ng/mL for triptonide (S/N = 3). The relative standard deviations of intra- and inter-day assay were less than 15% and the recoveries were better than 80%. The developed method was applied to the determination of triptolide and triptonide concentration in a patient's plasma after taking the medicament containing Tripterygium wilfordii Hook. F.     

A modified HPLC method for the determination of ochratoxin A by fluorescence detection

A high-performance liquid chromatographic method (HPLC) with fluorescent detector is described for the determination of ochratoxin A (OTA). A mobile phase consisting of acetonitrile:water:acetic acid (99:99:2, v/v/v) was used for the resolution of the compound on a C(18) Hypersil column. The retention time for OTA and diflunisal which was used as an internal standard (IS) were 11.7 and 12.8 min, respectively. The method is selective, reliable, reproducable with relative standard deviation (RSD) of 1.70 and linear in the range of 2.5 x 10(-9)-1.5 x 10(-8) M OTA. The limit of detection (LOD) and limit of quantification (LOQ) were 2.5 x 10(-10) M corresponding to 0.1 ng mL(-1) and 8.2 x 10(-10) corresponding to 3.3 ng mL(-1), respectively. Recovery studies were 81.2 +/- 1.9 (SD). The method was applied for analysis of OTA in wheat, corn, red pepper, cheese and wine. The proposed method can be used for the routine analysis of OTA in food and animal feed.     

高效液相色谱法测定水性酚醛树脂中残留的甲醛和苯酚

采用高效液相色谱法同时测定了水性酚醛树脂粘稠液体中残留的甲醛和苯酚,用2,4-二硝基苯肼对甲醛进行衍生化,然后进行色谱分离。色谱柱:Waters Symmetry C18 (5 μm,4.6 mm × 250 mm);柱温:30 ℃;流动相:甲醇-水(体积比为43∶57);检测波长:苯酚270 nm,甲醛360 nm;运行时间:45 min;流速:0.8 mL/min。方法的相对标准偏差小于5%,检测限小于1 μg/L,加标回收率为96%~105%。使用该分析方法可对该类树脂的质量性能进行有效监控。     

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CC(α)), detection capability (CC(β)), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples.     

High performance liquid chromatographic analysis of dehydroepiandrosterone and its pharmaceutical tablet formulation

A rapid, sensitive and stability indicating high performance liquid chromatographic method was developed and validated for the analysis of dehydroepiandrosterone (DHEA) in pharmaceutical tablet formulation. The analysis was done on a Supelcosil C(18) column (25 cm x 4.6 mm i.d., 5 microm). The mobile phase consisted of methanol:sodium acetate buffer solution (5 g/L):acetic acid (500 mL/L), 57:42:1, v/v/v, adjusted to pH 5 at a flow rate of 1 mL/min. Detection was carried out at a wavelength of 258 nm. The polynomial regression data for the calibration curve showed good linear relationship in the concentration range of 0.2-1 mg/mL with r = 0.9996. The method was validated for precision, accuracy and recovery. The limit of detection was found to be 50 ng/ microL. The method was applied for the analysis of DHEA in its pharmaceutical tablet formulation. The effects of different buffers and alcohols on the retention of DHEA were studied and the role of acetic acid as an organic phase modifier was also investigated.     

Simple determination of pirfenidone in rat plasma via high-performance liquid chromatography

A simple, rapid and reliable high-performance liquid chromatographic method was developed and validated for the determination of pirfenidone and its major metabolites in rat plasma. Plasma proteins were precipitated with perchloric acid (10%, v/v) and the supernatant after centrifugation was determined using high-performance liquid chromatography. The analysis was carried out on a Lichrospher C(18) column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water containing 0.2% acetic acid (23:77, v/v) at a flow-rate of 1 mL/min. The eluant was detected at 310 nm. The calibration curves were linear over a concentration range from 0.15 to 76.67 microg/mL. The accuracy (relative error) of the assay ranged from -2.6 to 7.9% and the precision (coefficient of variation) was less than 4.5%. The established method has been successfully applied to a pharmacokinetic study of pirfenidone following a single oral dose to rats.     

Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril-buffered aqueous solution containing 146 microL of triethylamine and 200 microL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine-N-oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine-N-oxide, respectively.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

Rapid determination of acetaminophen in physiological fluids by liquid chromatography using SDS mobile phase and ED detection

Acetaminophen is determined in serum and urine samples by a rapid, sensitive, and precise chromatographic method without any pretreatment step in a C18 column using a pure micellar mobile phase of 0.02M sodium dodecyl sulfate at pH 7. Acetaminophen is eluted in less than 5 min with no interference of the protein band. The use of electrochemical and UV detection is compared. Linearities (r > 0.999), as well as intra- and interday precision, are studied in the validation of the method. Limits of detection (LOD) are also calculated to be 0.56, 0.83, and 0.74 ng/mL in micellar solution, serum, and urine using electrochemical detection. The developed micellar liquid chromatographic method is useful for the quantitation of acetaminophen in serum and urine. Recoveries in the biological matrices are in the 98-107% range and results are compared with those obtained using a reference method. Drug excretion (in urine) and serum distribution are studied in several healthy volunteers, and no interference from metabolites is found. The developed procedure can be applied in routine analyses, toxicology, and therapeutic monitoring.