Development of a rapid method for the determination of glimepiride in human plasma using liquid-liquid extraction based on 96-well format micro-tubes and liquid chromatography/tandem mass spectrometry

A semi-automated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of glimepiride in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Glimepiride and the internal standard (IS) glibenclamide were extracted from human plasma by LLE, using a mixture of ethyl acetate/diethyl ether 50:50 (v/v) as the organic solvent. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analyte and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by reversed-phase LC/MS/MS with positive ion electrospray ionization, using multiple reaction monitoring. The method proved to be sensitive and specific for both drugs, and statistical evaluation revealed excellent linearity for the range of concentrations 2.0-500.0 ng/mL with very good accuracy and inter- and intra-day precisions. The proposed method enabled the rapid and reliable determination of glimepiride in pharmacokinetic or bioequivalence studies after per os administration of a 3 or 4 mg tablet of glimepiride.     

A rapid and sensitive liquid chromatography/positive ion tandem mass spectrometry method for the determination of cimetropium in human plasma by liquid-liquid extraction

We have developed and validated a simple detection system with high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) for determining cimetropium levels in human plasma using scopolamine butyl bromide as an internal standard (I.S.). The acquisition was performed in the multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 357.9 > 103.1 for cimetropium and m/z 359.9 > 103.1 for butyl-scopolamine. The method involves a simple single-step liquid-liquid extraction with dichloromethane. The analyte was chromatographed on an YMC C18 reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (19:81, v/v; adjusted to pH 4.0 with formic acid). The results were linear over the studied range (0.2-100 ng ml(-1)), with r2 = 1.0000, and the total analysis time for each run was 2 min. Intra- and interassay precisions were 0.70-8.54% and 1.08-4.85%, respectively, and intra- and interassay accuracies were 97.56-108.23% and 97.48-103.91%, respectively. The lower limit of quantification (LLOQ) was 0.2 ng ml(-1). At this concentration, mean intra- and interassay precisions were 8.54% and 4.85%, respectively, and mean intra- and interassay accuracies were 97.56% and 98.91%, respectively. The mean recovery ranged from 62.71 +/- 4.06 to 64.23 +/- 2.32%. Cimetropium was found to be stable in plasma samples under typical storage and processing conditions. The devised assay was successfully applied to a pharmacokinetic study of cimetropium bromide administered as a single oral dose (150 mg) to healthy volunteers.     

Dual negative precursor ion scan approach for rapid detection of glutathione conjugates using liquid chromatography/tandem mass spectrometry

Screening for conjugates formed by the tripeptide glutathione with new chemical entities is an essential step during the drug discovery process, as the formation of these conjugates serves as an indicator for the presence of reactive electrophilic intermediates. To increase the selectivity and throughput of this analysis, various mass spectral scan types have evolved over time. Historically, samples were analyzed under positive ionization conditions for the neutral loss of m/z 129 (loss of the pyroglutamic acid moiety from glutathione); however, more recently, negative precursor ion scanning for the loss of m/z 272 (deprotonated gamma-glutamyl-dehydroalanyl-glycine from glutathione) has emerged as a more selective tool. Further increasing the selectivity, we report on an extension of this methodology by incorporating a simultaneous dual negative precursor ion scan for two commonly observed ion fragments from glutathione conjugates, m/z 272 and 254 (the dehydrated form of m/z 272). This negative dual precursor ion scan methodology was first validated using substrates known to undergo metabolic bioactivation (diclofenac, carbamazepine, and 3-methyl indole) and has then been applied to the routine analysis of proprietary compounds undergoing active lead optimization. In comparison to alternative scan methodologies, the increased selectivity offered by this simultaneous dual precursor method results in a reduction in the generation of false positive results as well as reduced data interpretation time.     

A highly automated 96-well solid phase extraction and liquid chromatography/tandem mass spectrometry method for the determination of fentanyl in human plasma

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and fast liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, has been developed for the determination of the analgesic fentanyl in human plasma. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction (SPE) was carried out using a 96-channel programmable liquid-handling workstation using a mixed-mode sorbent. The extracted samples were then dried down, reconstituted and injected onto a silica column using an aqueous/organic mobile phase with tandem mass spectrometric detection. The method has been validated over the concentration range 0.05-100 ng/mL fentanyl in human plasma, based on a 0.25-mL sample size. The assay is sensitive, specific and robust. More than 2000 samples have been analyzed using this method. The automation of the sample preparation steps not only increased the analysis throughput, but also facilitated the transfer of the method between different bioanalytical laboratories of the same organization.     

Liquid-liquid extraction using 96-well plate format in conjunction with liquid chromatography/tandem mass spectrometry for quantitative determination of methylphenidate (Ritalin) in human plasma

Methylphenidate (MPH; Ritalin: methyl-alpha-phenyl-2-piperidinacetate hydrochloride) is utilized for the treatment of attention deficit hyperactivity disorder (ADHD) and narcolepsy. Recently, we described a rapid enantioselective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of the enantiomers of MPH (Rapid Commun. Mass Spectrom. 1999; 13: 2054). A lower limit of quantification (LLOQ) of 87 pg/mL was attained for the human plasma assay. The present paper describes a high-throughput sample preparation procedure in conjunction with racemic LC/MS/MS analysis for MPH with a LLOQ of 50 pg/mL. A semi-automated robotics method using liquid-liquid extraction (LLE) in a 96-well plate format was developed and validated. The correlation coefficients were > or =0.998 for MPH indicating good fits of the regression models over the range of the calibration curve. The accuracy and precision of the semi-automated approach were comparable to those obtained using the manual sample preparation technique reported previously (vide supra). The current method can easily be adapted to the enantioselective LC/MS/MS assay of MPH. The assay was simple, fast, specific, and exhibited excellent ruggedness.     

A rapid and sensitive liquid chromatography/tandem mass spectrometry method for determination of docetaxel in human plasma

A novel, rapid and sensitive isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for quantification of docetaxel in human plasma with paclitaxel as internal standard. The high sensitivity and specificity of MS/MS detection enabled the use of a small volume of plasma (0.05 mL) and a simple liquid-liquid extraction procedure. Furthermore, a very short run-time (3 min) fulfilled the need for monitoring plasma levels of docetaxel from large-scale clinical studies. The calibration curve for docetaxel was linear over the range 5-1000 ng/mL with coefficients of correlation >0.999 using only 0.05 mL plasma. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 96 to 110%. The applicability of the method was demonstrated in a pharmacokinetic study of a 1-h infusion of docetaxel with dosages of 75 mg/m(2). Possible conjugated metabolites of docetaxel were not detected in patients' samples.     

Simultaneous quantitation of enalapril and enalaprilat in human plasma by 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with rapid solid-phase extraction (SPE) has been developed and validated for the quantitative determination of enalapril and its active metabolite enalaprilat in human plasma. After addition of internal standard to human plasma, samples were extracted by 96-well SPE cartridge. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of enalapril and enalaprilat was accurate and reproducible, with respective limits of quantitation of 0.2 and 1.0 ng/mL in plasma. The standard calibration curves for both enalapril and enalaprilat were linear (r(2) = 0.9978 and 0.9998) over the concentration ranges 0.2-200 and 1.0-100 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for enalapril and enalaprilat were lower than 13.3 and 15.4% (relative standard deviation, %RSD), and accuracy was between 89.2-105.0 and 91.9-104.7%, respectively.     

Comparison of negative and positive ion electrospray tandem mass spectrometry for the liquid chromatography tandem mass spectrometry analysis of oxidized deoxynucleosides

Oxidized deoxynucleosides are widely used as biomarkers for DNA oxidation and oxidative stress assessment. Although gas chromatography mass spectrometry is widely used for the measurement of multiple DNA lesions, this approach requires complex sample preparation contributing to possible artifactual oxidation. To address these issues, a high performance liquid chromatography (HPLC)-tandem mass spectrometric (LC-MS/MS) method was developed to measure 8-hydroxy-2'-deoxyguanosine (8-OH-dG), 8-hydroxy-2'-deoxyadenosine (8-OH-dA), 2-hydroxy-2'-deoxyadenosine (2-OH-dA), thymidine glycol (TG), and 5-hydroxy-methyl-2'-deoxyuridine (HMDU) in DNA samples with fast sample preparation. In order to selectively monitor the product ions of these precursors with optimum sensitivity for use during quantitative LC-MS/MS analysis, unique and abundant fragment ions had to be identified during MS/MS with collision-induced dissociation (CID). Positive and negative ion electrospray tandem mass spectra with CID were compared for the analysis of these five oxidized deoxynucleosides. The most abundant fragment ions were usually formed by cleavage of the glycosidic bond in both positive and negative ion modes. However, in the negative ion electrospray tandem mass spectra of 8-OH-dG, 2-OH-dA, and 8-OH-dA, cleavage of two bonds within the sugar ring produced abundant S1 type ions with loss of a neutral molecule weighing 90 u, [M - H - 90]-. The signal-to-noise ratio was similar for negative and positive ion electrospray MS/MS except in the case of thymidine glycol where the signal-to-noise was 100 times greater in negative ionization mode. Therefore, negative ion electrospray tandem mass spectrometry with CID would be preferred to positive ion mode for the analysis of sets of oxidized deoxynucleosides that include thymidine glycol. Investigation of the fragmentation pathways indicated some new general rules for the fragmentation of negatively charged oxidized nucleosides. When purine nucleosides contain a hydroxyl group in the C8 position, an S1 type product ion will dominate the product ions due to a six-membered ring hydrogen transfer process. Finally, a new type of fragment ion formed by elimination of a neutral molecule weighing 48 (CO2H4) from the sugar moiety was observed for all three oxidized purine nucleosides.     

Liquid chromatography tandem mass spectrometry method for determination of N-acetylcysteine in human plasma using an isotope-labeled internal standard

A liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed to determine total N‐acetylcysteine in human plasma. Mass spectrometric detection was achieved in positive electrospray ionization and multiple reaction monitoring mode. The mass transition pairs of N‐acetylcysteine and the isotope‐labeled internal standard d3‐N‐acetylcysteine were 164 → 122 and 167 → 123, respectively. The method was linear over the range of 10–5000 ng/mL in human plasma. The adoption of trichloroacetic acid significantly enhanced the extraction recovery. The blank matrix was screened to minimize the influence of endogenous N‐acetylcysteine. After being fully validated, the method was successfully applied to the pharmacokinetic and bioequivalent study of N‐acetylcysteine after oral administration of 600 mg tablets to 24 healthy Chinese volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

A sensitive method for determination of allergenic fragrance terpene hydroperoxides using liquid chromatography coupled with tandem mass spectrometry

Different compositions of monoterpenes are utilized for their pleasant scent in cosmetics and perfumes. However, the most commonly used fragrance terpenes easily oxidize upon contact with air, forming strongly skin‐sensitizing hydroperoxides. Due to their thermolability and low UV absorbance, detection methods for hydroperoxides are scarce. For the first time, a simple and sensitive method using LC/ESI‐MS/MS was developed to quantitatively determine hydroperoxides from the common fragrance compounds linalool, linalyl acetate, and limonene. The method was applied to autoxidized petitgrain oil and sweet orange oil. A separation was accomplished using a C₃ column. The method LOD for the investigated hydroperoxides in the essential oils was below 0.3 μg/mL, corresponding to 0.3 ppm. For prevention purposes and according to EU regulations, concentrations in cosmetics exceeding 100 ppm in “rinse‐off” and 10 ppm in “stay‐on” products of linalool and limonene must be labeled. However, the products may still contain allergens, such as hydroperoxides, formed by oxidative degradation of their parent terpenes. The sensitivity and selectivity of the presented LC/MS/MS method enables detection of hydroperoxides from the fragrance terpenes linalool, linalyl acetate, and limonene. However, for routine measurements, the method requires further validation.     

Quantification of pramipexole in human plasma by liquid chromatography tandem mass spectrometry using tamsulosin as internal standard

A high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantification of pramipexole in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H](+) ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited a linear dynamic range of 200-8000 pg/mL for pramipexole in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 200 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Comparison of turbulent-flow chromatography with automated solid-phase extraction in 96-well plates and liquid-liquid extraction used as plasma sample preparation techniques for liquid chromatography-tandem mass spectrometry.

Turbulent flow chromatography (TFC) combined with the high selectivity and sensitivity of tandem mass spectrometry (MS-MS) is a new technique for the fast direct analysis of drugs from crude plasma. TFC in the 96-well plate format reduces significantly the time required for sample clean-up in the laboratory. For example, for 100 samples the workload for a technician is reduced from about 8 h by a manual liquid-liquid extraction (LLE) assay to about 1 h in the case of TFC. Sample clean-up and analysis are performed on-line on the same column. Similar chromatographic performance and validation results were achieved using HTLC Turbo-C18 columns (Cohesive Technologies) and Oasis HLB extraction columns (Waters). One 96-well plate with 96 plasma samples is analyzed within 5.25 h, corresponding to 3.3 min per sample. Compared to this LLE and analysis of 96 samples takes about 16 h. Two structurally different and highly protein bound compounds, drug A and drug B, were analyzed under identical TFC conditions and the assays were fully validated for the application to toxicokinetics studies (compliant with Good Laboratory Practices-GLP). The limit of quantitation was 1.00 microg/l and the linear working range covered three orders of magnitude for both drugs. In the case of drug A the quality of analysis by TFC was similar to the reference LLE assay and slightly better than automated solid-phase extraction in 96-well plates. The accuracy was -3.1 to 6.7% and the precision was 3.1 to 6.8% in the case of drug A determined for dog plasma by TFC-MS-MS. For drug B the accuracy was -3.7 to 3.5% and the precision was 1.6 to 5.4% for rat plasma, which is even slightly better than what was achieved with the validated protein precipitation assay.     

Liquid chromatography/electrospray tandem mass spectrometry method for the quantitation of fosinoprilat in human serum using automated 96-well solid-phase extraction for sample preparation

A sensitive, specific, accurate and reproducible liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the quantitation of fosinoprilat in 0.2 mL of human serum. The method employed acidification (with pH 4.0 sodium acetate buffer) of the serum samples to minimize the hydrolysis of the prodrug fosinopril to fosinoprilat prior to purification by automated 96-well solid-phase extraction. The required chromatographic separation of fosinoprilat and fosinopril was achieved isocratically on a Luna C8 analytical column (2 x 50 mm, 3 microm). The total run time was 2 min. The mobile phase contained methanol and water with 10 mM ammonium acetate. Detection was by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 2.00 to 500 ng/mL, was fitted to a 1/x(2) weighted linear regression model. Fosinoprilat quality control (QC) samples used to determine the accuracy and precision of the method were prepared in human serum at concentrations of 5.00, 200, 400 and 1000 ng/mL. The assay accuracy was within 8% (dev). The intra- and inter-assay precisions were within 6 and 3% (RSD), respectively. Fosinopril QC samples used to gauge the rate of hydrolysis of fosinopril to fosinoprilat during the assay procedure were prepared in human serum at 500 ng/mL. The hydrolysis of fosinopril to fosinoprilat was 相似文献   

Enantiomeric separation and quantification of fluoxetine (Prozac) in human plasma by liquid chromatography/tandem mass spectrometry using liquid-liquid extraction in 96-well plate format

Fluoxetine (Prozac) is currently one of the widely prescribed selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression. A high-throughput sample preparation procedure using liquid-liquid extraction (LLE) in a 96-well plate format in conjunction with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for quantification of fluoxetine enantiomers in human plasma. After addition of internal standard and ammonium hydroxide, samples were extracted with ethyl acetate. The organic extract was evaporated to dryness and reconstituted in methanol. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Adequate separation of fluoxetine enantiomeric pairs (resolution of 1.17) was achieved on a vancomycin column eluted with methanol containing 0.075% (by weight) ammonium trifluoroacetate. A triple quadrupole mass spectrometer, operated in the multiple reaction monitoring mode at m/z 310-->44 for fluoxetine enantiomeric pairs and m/z 287-->241 for oxazepam (internal standard), was used. Analysis was performed in the positive ion mode using atmospheric pressure chemical ionization (APCI). The standard curve range was 2.0-1000 ng/mL for each fluoxetine enantiomer. The intra- and inter-day precision and accuracy of the quality control (QC) samples were <12.5% (CV) and <13.6% (CV), respectively, for each fluoxetine enantiomer; the correlation coefficient was >0.990. Method ruggedness was demonstrated by the reproducible performance of the assay during a three-day validation period.     

Workplace monitoring of isocyanates using ion trap liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/ion trap tandem mass spectrometry method was developed for the qualitative and quantitative detection of isocyanates in air. The method is based on derivatization of isocyanates with 1-(2-methoxyphenyl)piperazine during air sampling. The extracts are analyzed using an ion trap LC/MS system equipped with an electrospray (ESI) ion source. The method shows high linearity, specificity, accuracy and precision. The limits of detection are 40x to 55x lower than with UV-based methods.     

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.     

Method development for the concentration determination of a protein in human plasma utilizing 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometric detection

Although liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology has been widely used for quantitative analysis of small organic molecules, it has been a challenging task to quantitatively analyze protein samples utilizing this technology in biological matrices for pre-clinical and clinical studies. Here we present our initial results in method development for the quantitative determination of rK5 protein concentrations in human plasma samples utilizing LC/MS/MS technology. A protein similar in structure to rK5, but with a slightly reduced molecular weight, was used as internal standard. A 96-well solid-phase extraction procedure was developed to effectively extract protein analytes from plasma samples. Quantitative analysis was obtained by a novel approach of protein monitoring that employed selective reaction monitoring (SRM). Even though mass spectrometry of the internal standard protein gave no fragment ions, SRM monitoring greatly reduced background interference. Using samples prepared in human plasma with sodium EDTA as anticoagulant, a correlation coefficient (r(2)) of 0.9940 was obtained by producing a single standard curve with the injection of six rows of standards with a concentration range from 100 ng/mL to 10 microg/mL. The mean analytical recovery for these standards ranged from 91.5 to 103.6%. The CVs for individual standard levels ranged from 3.7 to 20.9%. The experiment was also repeated using samples prepared in human plasma with sodium heparin as anticoagulant, which produced a correlation coefficient (r(2)) of 0.9952 obtained from a single standard curve with the injection of four rows of standards with a concentration range from 50 ng/mL to 10 microg/mL. The mean analytical recovery for the standards ranged from 96.2 to 104.6%. The CVs for individual standard levels ranged from 2.6 to 15.6%.     

Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma

A sensitive, rapid and specific quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of apomorphine (APO) in canine plasma. The analytes were prepared using one-step liquid-liquid extraction, and analyzed on a Waters Symmetry C(18) column interfaced with triple quadrupole tandem mass spectrometer. A mixture of methanol/0.1% formic acid in water (70: 30, v/v) was employed as the isocratic mobile phase. Positive electrospray ionization was utilized as the ionization source. The analyte and clenbuterol (internal standard) were both detected using multiple reaction monitoring (MRM) mode. The limit of detection (LOD) obtained was 0.03 ng/mL. The assay was linear over the concentration range of 0.1-100 ng/mL, and provided good precision (RSD) and good accuracy (RE). The analyte was stable by using antioxidants throughout the whole study. The experimental results show that LC/MS/MS is a rapid and sensitive method to analyze APO in plasma. Finally, the proposed method was successfully applied to a pharmacokinetic study of APO after intranasal administration of 0.5 mg apomorphine to 10 healthy beagle dogs.     

High-throughput sample preparation and analysis using 96-well membrane solid-phase extraction and liquid chromatography-tandem mass spectrometry for the determination of steroids in human urine

A 96-well solid-phase extraction (SPE) system is used to rapidly prepare human urine samples for high-throughput quantitative analysis of two steroids, equilenin and progesterone, by liquid chromatography-tandem mass spectrometry using deuterated estrone as the internal standard. We define high-throughput here as analysis of 384 samples in a 24 h period. A total of 384 samples and standards were extracted by an individual in one day and subsequently analyzed within a 24 h period. The inter- and intratray accuracy and precision obtained over the course of these injections was within 8% coefficient of variation when analyzed by atmospheric pressure chemical ionization mass spectrometry using positive ion detection. A semiautomated sample processing workstation was used to add internal standard and then process 96 samples at a time. The recovery of the analytes from the SPE was approximately 85%. The accuracy and precision obtained was comparable to that ordinarily obtained using manual sample preparation techniques.     

An improved high-throughput liquid chromatographic/tandem mass spectrometric method for terbinafine quantification in human plasma, using automated liquid-liquid extraction based on 96-well format plates

A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for terbinafine quantification in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2 mL 96-deepwell plates. Terbinafine and the internal standard (IS) N-methyl-1-naphthalenemethylamine were extracted from human plasma by LLE, using a mixture of methyl t-butyl ether (MTBE)-hexane (70:30, v/v) as the organic solvent. All liquid transfer steps, including preparation of calibration standards and quality control samples, as well as the addition of the IS, were performed automatically by using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of a reconstitution solution. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The method had a very short sample preparation time and a chromatographic run time of 2.2 min. It was proved to have excellent sensitivity, specificity, accuracy as well as inter- and intraday precision for the quantification of terbinafine in human plasma. The calibration curve was linear for the range of concentrations 5.0-2000.0 ng/mL. The proposed method was applied to the rapid and reliable determination of terbinafine in a bioequivalence study after per os administration of 250 mg tablet formulations of terbinafine.