Clonazepam quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method for quantifying clonazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a hexane/diethylether (20 : 80, v/v) solution. The extracts were analysed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on a Jones Genesis C8 4 microm analytical column (100 x 2.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 0.5-50 ng/ml (r2 > 0.9965). The limit of quantification was 0.5 ng/ml. This HPLC/MS/MS procedure was used to assess the bioequivalence of two clonazepam 2 mg tablet formulations (clonazepam test formulation from Ranbaxy Laboratories Ltd and Rivotril from Roche Laboratórios Ltda as standard reference formulation).     

血、尿中氯硝西泮及其代谢物7-氨基氯硝西泮的GC-ECD法检测

报道了血、尿中氯硝西泮及其代谢物7－氨基氯硝西泮的GC－ECD检测方法。苯－异戊醇碱性条件下（pH10.8）液－液萃取，灵敏度较高，氯硝西泮和7－氨基氯硝西泮的检测限（LOD）分别为3．2ng/mL及1．7ng/mL。线性范围5－300ng/mL，RSD5．3％。     

Efficient HPLC method for determination of cephalosporin residues on spiked stainless-steel plates and human plasma: application of a worst-case product for Cosa®CIP

ABSTRACT

The main objective of the cleaning validation procedure is to verify the effectiveness of the cleaning procedure for removal and minimising the risk of cross-contamination, a topic that has become more important regarding the development of the medicines. Furthermore, if a product is found to be the worst among many of the products, one cleaning validation procedure of the worst-case product can cover the validation of the remaining ones, thus saving time and money. A novel, reproducible and efficient high-performance liquid chromatography (HPLC) method was optimised and validated for the detection of the following cephalosporin residues: cephalexin (CPH), cefaclor (CFC), cefixime (CFX), cefdinir (CFR) and ceftazidime (CFZ) in human spiked plasma and in production machines using rinse and swab sampling collected from surfaces and application to Cosa®CIP Detergent. Isocratic chromatographic system was performed at ambient temperature using mobile phase consisting of acetonitrile: 40% tetrabutylammonium hydroxide adjusted to pH 7.0 ± 0.1 with 10% phosphoric acid (72.5:27.5, v/v) on Ultrasphere ion pair column (250 mm × 4.6 mm, 5.0 μm particle size) at a flow rate 1.0 mL/min, injection volume 10 μL and UV detection at 265 nm. The chromatographic run time was less than 20 min for the mixture. Linear relationships were obtained over the concentration ranges 0.5–25 ng mL^?1 for CPH, 1.5–30 ng mL^?1 for CFC, 2–33 ng mL^?1 for CFX, 3–35 ng mL^?1 for CFR and 4–40 ng mL^?1 for CFZ with correlation coefficients >0.998. Analytical and bioanalytical validation methods were carried out following terms of linearity, specificity, LOQ, LOD, accuracy and precision for determination of cephalosporin residues in production machines and in human spiked plasma according to FDA guidelines.     

Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10--15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.     

高效薄层法检测尿中氟硝西泮的代谢物7-氨基氟硝西泮

 报道了尿中氟硝西泮代谢物 7 氨基氟硝西泮 (7AFLZ)的高效薄层 (HPTLC)定性分析和半定量分析方法 ,分析物斑点用荧光胺进行荧光显现 ,灵敏度高 ,检测限 (LOD)为 5 μg/L ,测量限 (LOQ)为 15 μg/L。该方法可以检测人口服 1mg氟硝西泮后 48h内排泄尿中的 7AFLZ ,适于麻醉抢劫犯罪案件中的药物检测。     

Validation of SPE‐HPLC determination of 1,4‐benzodiazepines and metabolites in blood plasma,urine, and saliva

A simple, sensitive, selective, and reproducible RP‐HPLC method with DAD detection at 240 nm was developed for the determination of six 1,4‐benzodiazepines: bromazepam (BRZ), clonazepam (CLZ), diazepam (DZP), flunitrazepam (FNZ), lorazepam (LRZ), alprazolam (APZ); and two metabolites: α‐hydroxyalprazolam (HALZ) and α‐hydroxytriazolam (HTZL) in human plasma, urine, and saliva, using colchicine as internal standard, after SPE using Nexus Varian cartridges. Separation was performed on a Kromasil C₈ (250 mm×5 mm, 5 μm) analytical column with a gradient mobile phase containing methanol, ACN and 0.05 M ammonium acetate. Linearity was held within the range 0.3–20.0 ng/μL, with coefficients of determination (r²) better than 0.997. The within‐ and between‐day assay RSD at 2, 4, 8 ng/μL ranged from 0.03 to 4.7% and 0.5 to 7.0%, respectively in standards, from 1.3 to 7.9% and 3.3 to 7.3%, respectively in plasma, from 2.1 to 6.0% and 2.1 to 7.8%, respectively in urine and at 0.5, 1.0, 2.0 ng/μL ranged from 2.22 to 5.8% and 2.2 to 8.1%, respectively, in saliva. The mean relative recoveries were 96.3–108.6, 96.0–108.2, 94.3–107.1, 97.0–107.0% in within‐day assay and 96.8–107.7, 94.6–107.6, 93.2–105.8, 96.0–108.6 in between‐day assay for standard, plasma, urine, and saliva, respectively. The LOD and LOQ were 0.02–0.47 and 0.07–1.57 ng/μL, respectively.     

LC–MS Method for the Quantification of Clonazepam in Rat Plasma

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of clonazepam in rat plasma. Clonazepam and internal standard diazepam were extracted from plasma samples by a single-step protein precipitation. The chromatographic separation was performed on a Dikma ODS-C18 reversed-phase column at 40 °C. The mobile phase composed of a premix of solvent A (0.1% formic acid–4 mM ammonium acetate–water)–solvent B (acetonitrile) (13:87, v/v) at a flow-rate of 0.7 mL min^?1. Positive electrospray ionization was utilized as the ionization source. Clonazepam and the internal standard were determined using multiple reaction monitoring of precursor → product ion transitions at m/z 316.0 → 270.0 and m/z 285.1 → 193.2, respectively. The lower limit of quantification was 0.25 ng mL^?1 using 50 μL plasma samples and the linear calibration range was from 0.25 to 128 ng mL^?1. The within- and between-batch RSDs were lower than 15% and the relative recoveries of clonazepam ranged from 97.4 to 104.7%. The mean extraction recoveries of clonazepam and IS were 79.7 and 77.6%, respectively. The method has been successfully applied to the pharmacokinetic studies in rat after oral administration of clonazepam.     

High-Performance Liquid Chromatographic Determination in Human Plasma of a Anticonvulsant Benzodiazepine: Clonazepam

Abstract

A rapid and specific high-pressure liquid chromatography method for determination of clonazepam in human plasma is described.

The analysis is linear for concentrations ranging from 5 to 100 ng. ml^-1 plasma for clonazepam.

The method is applicable to quantitation of clonazepam in human plasma of subjects receiving 0.05 at 0.20 mg.kg^-1 orally, with satisfactory accuracy and precision.     

Adsorptive Stripping Differential Pulse Voltammetry for Determination of Trace Amounts of Noscapine in Human Plasma

A new adsorptive anodic differential pulse stripping voltammetry method for the direct determination of noscapine at trace levels in human plasma of addicts is proposed. The procedure involves an adsorptive accumulation of noscapine on a hanging mercury drop electrode (HMDE), followed by oxidation of adsorbed noscapine by voltammetry scan using differential pulse modulation. The optimum conditions for the analysis of noscapine are pH = 8.5 using Britton‐Robinson (B‐R) buffer, accumulation potential of ?100 mV (vs. Ag/AgCl), and accumulation time of 150 s. The peak current is proportional to the concentration of noscapine, and a linear calibration graph is obtained at 0.015–2.75 μg mL^?1. A relative standard deviation of 1.28% (n = 5) was obtained, and the limit of detection was 7 ng mL^?1. The capability of the method for the analysis of real samples was evaluated by determination of noscapine in spiked human plasma and addicts, human plasma with satisfactory results.     

Determination of Organotin Compounds in Wine by Microwave-Assisted Extraction and High Performance Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry

A new analytical procedure for the determination of five organotin compounds in several matrix wine samples is reported. The organotin compounds were extracted by microwave-assisted extraction with n-hexane. Extraction conditions, such as volume of n-hexane required, extraction temperature, and extraction time, were investigated and optimized by an orthogonal array experimental design. The determination of organotin compounds in the final extracts was carried out by liquid chromatography–inductively coupled plasma mass spectrometry. The procedure showed limits of detection between 0.029–0.049 µg · L^?1. The linearity was in the range of 0.5 to 100 µg · L^?1. The precision expressed as relative standard deviation (RSD) was below 9.43%. The developed method was successfully employed to analyze different matrix wine samples, and some analytes were detected at the level of 0.053 to 1.14 µg · L^?1.     

Simultaneous HPTLC Determination of Escitalopram Oxalate and Clonazepam in Combined Tablets

A new, simple high-performance thin-layer chromatographic method has been established and validated for simultaneous determination of escitalopram oxalate and clonazepam in a combined tablet dosage form. The drugs were separated on aluminum plates precoated with silica gel 60 F₂₅₄; toluene–ethyl acetate–triethylamine 7:3.5:3 (v/v) was used as mobile phase. Quantitative analysis was performed by densitometric scanning at 258 nm. The method was validated for linearity, accuracy, precision, and robustness. The calibration plot was linear over the ranges 250–2,500 and 50–500 ng band⁻¹ for escitalopram oxalate and clonazepam, respectively. The method was successfully applied to the analysis of drugs in a pharmaceutical formulation.     

Rapid Determination of Montelukast in Human Plasma by LC-ESI-MS/MS and Its Application to a Bioequivalence Study

Abstract

A rapid liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the determination of montelukast in human plasma. The extraction of montelukast from plasma (300 µL) involved protein precipitation. Quantitation was performed using LC-ESI-MS/MS, operating in the positive ion and selective reaction monitoring (SRM) mode. The total chromatographic run time for the analysis was 1.5 min. A linear dynamic range was established from 5 to 800 ng mL^?1 for montelukast. The method was fully validated especially with regard to real subject sample analysis. It was successfully applied to a bioequivalence study in 18 healthy human subjects under fed condition (human subjects were allowed to eat as per the prescribed diet for bioequivalence study).     

Determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma by high-performance liquid chromatography and fluorescence detection

A high-performance liquid chromatographic method for the simultaneous determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma is described. After extraction with an organic solvent, the compounds and their internal standard (7-amino-methylclonazepam or Ro 5-3384) are derivatized with fluorescamine and chromatographed on a reversed-phase muBondapak C18 column using pH 8 buffer solution-acetonitrile (3:1) as mobile phase. The detection is performed by a fluorometer at excitation and emission wavelengths of 390 and 470 nm, respectively. The sensitivity limit is about 1 ng/ml of plasma for both 7-amino-flunitrazepam and 7-amino-desmethylflunitrazepam. The method has been applied to the determination of plasma levels of these substances during pharmacokinetic studies of flunitrazepam, desmethylflunitrazepam and 7-amino-flunitrazepam.     

Determination of Fluoxetine in Human Plasma by Liquid Chromatography–Mass Spectrometry and Its Application

Abstract

A rapid, simple, and specific liquid chromatography–electrospray ionization–mass spectrometric method has been developed and validated for the determination of fluoxetine in human plasma. The method was validated with a linear range of 0.5–100 ng mL^?1, and the lowest limits of quantification were 0.5 ng mL^?1 for fluoxetine. The extraction efficiencies were about 65% and recoveries of method were in the range of 94.0–97.5%. The intraday relative standard deviation (RSD) was less than 11% and interday RSD was within 12%. The method has been successfully applied to the evaluation of pharmacokinetics and bioequivalence of fluoxetine.     

Determination of the neuroprotective agent 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione in plasma using high-performance liquid chromatography.

A rapid, sensitive and selective high-performance liquid chromatographic method for the determination of the neuroprotectant 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione in rat plasma has been established and validated. Samples of 0.5 ml of plasma are extracted by elution from a Bond-Elut column with methanol and analysed on a reversed-phase column. The wavelength of UV detection is 254 nm. The method is linear at least up to 30 micrograms/ml, with a lowest reliable determination level of 4 mg/ml. The assays has a coefficient of variation of 13% at 10 ng/ml and 4% at 1000 ng/ml. Small variations in the extraction procedure and the liquid chromatographic conditions have minimal or no influence on the assay.     

Determination of Ferulic Acid in Rat Plasma by Liquid Chromatography–Tandem Mass Spectrometry Method: Application to a Pharmacokinetic Study

Abstract

A rapid, sensitive, and specific liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method has been developed for quantification of ferulic acid in rat plasma. The analyte and docetaxel (internal standard) were extracted from plasma samples with diethyl ether and analyzed on a C₁₈ column. The chromatographic separation was achieved within 3.5 min by using acetonitrile-water as the mobile phase and the flow rate was 0.2 mL · min^?1. The method was linear within the range of 0.5 ? 800 ng · mL^?1. The lower limit of quantification (LLOQ) was 0.5 ng · mL^?1. Finally, the method is successfully applied for the pharmacokinetic study of ferulic acid in rats following intravenous administration.     

Thin-layer chromatographic separation and in situ fluorimetric determination of ofloxacin in plasma and pleural fluid

A thin-layer chromatographic assay for the determination of ofloxacin in human plasma and pleural fluid is described. After extraction of ofloxacin from samples with dichloromethane, chromatography was performed on thin-layer plates (silica gel) with a mobile phase consisting of ethanol and water; the tank atmosphere was equilibrated with concentrated ammonia. The precision of the assay could be considerably increased along with the measured fluorescence intensity of ofloxacin by spraying the plate with a citric acid solution and dipping it into paraffin or using a mixture of both components. Peaks were quantified by densitometric evaluation of the chromatograms. The method shows a very low limit of detection (1 ng/ml) as well as good precision and linearity in the range 0.001-2.0 micrograms/ml for both plasma and pleural fluid.     

High-Performance Liquid Chromatographic Assay for Chlorquine and its Two Major Metabolites,Desethylchloroquine and Bidesethylchloroquine in Biological Fluids

Abstract

A high-performance liquid chromatography (HPLC) method for the determination of chloroquine and its two major metabolites in biological fluids is described. Hydroxychloroquine is used as an internal standard (I.S.). Drug, metabolites and I.S. were extracted as bases with diethyl ether by a single step procedure. After drying and evaporation of the organic phase, the residue was dissolved into the mobile phase and injected into the chromatographic system. Separation was performed using a normal phase column (Inertsil sill with mixture of acetonitrile, methanol and ammonia as mobile phase. The detection was carried out by fluorescence measurement : excitation wavelength was set at 325 nm and emission at 380 nm. The limit of detection was near 3.7 ng ml^?1 for chloroquine and metabolites. No chromatographic interference could be detected by endogenous compounds or other antimalarial drugs. Because of the good accuracy of the method, concentrations were determinated with a relative standard deviation lower than 7% at the 25 ng ml^?l level for all substances.

An excellent precision was obtained over the range of concentrations tested, 25–1000ng ml^?l. This method can be applied to therapeutic, pharmacokinetic and epidemial studies.     

胶束液相色谱法同时测定血浆中的苯巴比妥、艾司唑仑和氯硝西泮

采用胶束液相色谱法(MLC)分离测定血浆中苯巴比妥、艾司唑仑和氯硝西泮,运用三相平衡理论探讨了流动相中表面活性剂浓度(CM)、氢离子浓度(CH)、助表面活性剂浓度(Cφ)对溶质保留行为的影响,同时运用多元线性回归建立了保留因子的对数(log k)与溶质性质参数和流动相组成之间的相关模型。结果表明,溶质保留因子(k)随CM、Cφ和CH的增加而减小,与理论模型完全一致。而且log k与溶质的疏水性常数的对数(log P)和电离常数(Ka)以及CM、CH和Cφ之间呈现良好的多元线性关系。在确定的色谱条件下,血浆中的3种药物与其他组分之间有较好的分离效果。3种药物的血药浓度分别在2.5～50 mg/L、0.25～5.0 mg/L和0.05～5.0 mg/L间具有良好的线性关系。本方法简便、准确、重现性良好、灵敏度高。3种药物的最低检出限(S/N=3)分别为10.27、1.17、0.867 ng,平均加标回收率范围分别为99.80%～102.9%, 94.00%～98.20%和96.30%～98.70%。     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.