A clinical trial on a plate? The potential of 384-well format solid phase extraction for high-throughput bioanalysis using liquid chromatography/tandem mass spectrometry

The application of 384-well format solid phase extraction (SPE) for bioanalysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) is reported and a 384-well SPE method for the 5-HT agonist sumatriptan in human plasma described. Plasma samples were extracted on a prototype low-density polyethylene 384-well SPE block using a packed bed of 5 mg Oasistrade mark HLB. Liquid handling was automated by a combination of a robotic sampler processor and a 96/384 multi-channel dispensing station. Samples and SPE reagents were drawn through the SPE block by centrifugation. The extracts were analysed by LC/MS/MS with thermally and pneumatically assisted electrospray ionisation and selected reaction monitoring. The method is used to illustrate and discuss the feasibility and viability of sample preparation techniques in high-density microtitre plate format for routine bioanalysis.     

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.     

Trapping 4-fluorobenzyl chloride in human plasma with chemical derivatization followed by quantitative bioanalysis using high-performance liquid chromatography/tandem mass spectrometry

A quantitative bioanalytical method involving chemical derivatization, solid phase extraction (SPE) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was developed for the determination of 4-fluorobenzyl chloride (4FBCl) in human plasma. 4FBCl is a volatile and reactive molecule that is very unstable in human plasma. In order to stabilize 4FBCl in plasma samples prior to storage, 4-dimethylaminopyridine (DMAP) was added, forming a stable quaternary amine salt derivative. A three-step weak cation-exchange SPE procedure was then employed to remove excess DMAP. The plasma extracts were analyzed by HPLC/MS/MS using a TurboIonspray interface and multiple reaction monitoring. Unlike 4FBCl, the quaternary amine derivative shows excellent sensitivity in electrospray mass spectrometry. The method was validated over a concentration range of 0.5-500 ng/mL using 45 microL of plasma. The maximum within-run and between-run precision observed in a three-run validation for quality control (QC) samples was 12.5 and 7.6%, respectively. The maximum percentage bias observed at all QC sample concentrations was 11.9%. The method has proven to be robust and compatible with high-throughput bioanalysis.     

High-speed gradient parallel liquid chromatography/tandem mass spectrometry with fully automated sample preparation for bioanalysis: 30 seconds per sample from plasma

In this work, a high-throughput and high-performance bioanalytical system is described that is capable of extracting and analyzing 1152 plasma samples within 10 hours. A Zymark track robot system interfaced with a Tecan Genesis liquid handler was used for simultaneous solid-phase extraction of four 96-well plates in a fully automated fashion. The extracted plasma samples were injected onto four parallel monolithic columns for separation via a four-injector autosampler. The use of monolithic columns allowed for fast and well-resolved separations at a considerably higher flow rate without generating significant column backpressure. This resulted in a total chromatographic run cycle time of 2 min on each 4.6 x 100 mm column using gradient elution. The effluent from the four columns was directed to a triple quadrupole mass spectrometer equipped with an indexed four-probe electrospray ionization source (Micromass MUX interface). Hence, sample extraction, separation, and detection were all performed in a four-channel parallel format that resulted in an overall throughput of about 30 s per sample from plasma. The performance of this system was evaluated by extracting and by analyzing twelve 96-well plates (1152) of human plasma samples spiked with oxazepam at different concentrations. The relative standard deviation (RSD) of analyte sensitivity (slope of calibration curve) across the four channels and across the 12 plates was 5.2 and 6.8%, respectively. An average extraction recovery of 77.6% with a RSD of 7.7% and an average matrix effect of 0.95 with a RSD of 5.2% were achieved using these generic extraction and separation conditions. The good separation efficiency provided by this system allowed for rapid method development of an assay quantifying the drug candidate and its close structural analog metabolite. The method was cross-validated with a conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay.     

Increasing the efficiency of pharmacokinetic sample procurement, preparation and analysis by liquid chromatography/tandem mass spectrometry

The movement towards a 96-well format has greatly increased productivity and throughput in bioanalytical laboratories. Improvements in automated sample preparation and analytical methods have further contributed to increased productivity. We have focused on sample collection and transfer to the bioanalyst and have found improvements to the current available methods. The problem of manual transfers and plasma clotting issues can be overcome with the use of microtainers. Specifically, for illustrative purposes, three proprietary Theravance compounds were tested for stability, non-specific binding, and electrospray ion suppression in microtainers. There were no issues with stability, non-specific binding or ion suppression for the above compounds even after leaving plasma samples in the microtainers over long periods of time. The microtainers are robot-compatible and the resulting plasma can be transferred without clotting issues. To date, all in-house compounds successfully analyzed and tested using the microtainers have mass ranges between 200 and 1800 Da, pK(a) ranges between 3.8 and 10.3, and logD ranges between -1.7 and 4.2. Once samples are transferred into 96-well plates, flexibility in preparation and analysis is available. Together with automated sample preparation and the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an analytical tool, the use of microtainers as sample collection tubes and for sample storage saved considerable time, cost and effort in both of our pharmacokinetic (PK) and bioanalytical groups. This in turn has led to an increased efficiency and overall throughput in support of our drug discovery effort.     

Development and validation of amitriptyline and its metabolite in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study

A method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) in combination with solid‐phase extraction for sample pretreatment has been developed for the simultaneous analysis of amitriptyline and its main metabolite in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective SPE procedure using hydrophilic–lipophilic balance cartridges. The assay involves a simple solid‐phase extraction (SPE) procedure of 0.2 mL of human plasma and analysis was performed on a triple‐quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode via electrospray ionization (ESI). The standard calibration curve was linear over the ranges 0.370–95.539 ng/mL for amitriptyline and 0.365–94.374 ng/mL for nortriptyline, expressed by the linear correlation coefficient r², which was better than 0.995 for both. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recovery was 85.3, 88.4 and 80.7% for amitriptyline, nortriptyline and doxepin respectively. Total run time was 1.2 min only for each sample, which makes it possible to analyze more than 400 samples per day. The method was highly reproducible and gave peaks with excellent chromatography properties. Copyright © 2010 John Wiley & Sons, Ltd.     

A strategy for liquid chromatography/tandem mass spectrometry based quantitation of pegylated protein drugs in plasma using plasma protein precipitation with water-miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection

Recently, we have developed liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods for the quantitation of pegylated therapeutic proteins in plasma. The methods are based on the LC/MS/MS detection of a surrogate peptide generated from trypsin digestion of the therapeutic protein. Various parameters related to the bioanalytical methods were evaluated and optimized, including the preparation of calibration standards and quality control samples, sample extraction, internal standard selection and its stage of addition, trypsin digestion, and non-specific binding. In this paper, we report the development of a method for a specific pegylated therapeutic protein and detail the various optimization steps undertaken. Simple extraction of the pegylated therapeutic protein from plasma was achieved via the precipitation of the endogenous proteins in plasma using acidic isopropanol and the resulting supernatant extract was subjected to trypsin digestion. A unique tryptic peptide arising from the pegylated therapeutic protein was used for LC/MS/MS-based detection and quantitation. A protein and a peptide were used as internal standards, with the former added before the sample extraction and the latter after the sample extraction. The method developed is simple, sensitive, specific and rugged, and has been implemented in a high throughput 96-well format to analyze plasma samples from in vivo studies. A required lower limit of quantitation (LLOQ) of 10 ng/mL, expressed in terms of the concentration of the protein drug, was easily achieved.     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

A high‐throughput bioanalytical platform using automated infusion for tandem mass spectrometric method optimization and its application in a metabolic stability screen

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate‐based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high‐quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan? 2.0, a unique high‐throughput solution featuring MS/MS method optimization by automated infusion, with a customized in‐house software tool in support of a Metstab screen. In this platform, a dual‐column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of a 'GW cocktail' of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry

A mass spectrometry based method for the simultaneous determination of an in vivo Greenford-Ware or 'GW cocktail' of CYP450 probe substrates and their metabolites in both human plasma and urine is described. The probe substrates, caffeine, diclofenac, mephenytoin, debrisoquine, chlorzoxazone and midazolam, together with their respective metabolites and stable isotope labelled internal standards, are simultaneously extracted from the biological matrix using solid phase extraction in 96-well microtitre plate format, automated by means of a custom built Zymark robotic system. The extracts are analysed by fast gradient high performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (MS/MS) using thermally and pneumatically assisted electrospray ionisation in both positive and negative ion modes and selected reaction monitoring. The methods are specific, accurate and precise with intra- and inter-assay precision (%CV) of less than 15% for all analytes.     

Development and validation of automated SPE‐LC‐MS/MS method for determination of indapamide in human whole blood and its application to real study samples

A fast and simple liquid chromatography–electrospray ionization tandem mass spectrometry method for determination of indapamide in human whole blood was developed and validated. The sample extraction of indapamide from human whole blood was achieved using automated solid‐phase extraction. Chromatographic separation was performed on Kinetex C₁₈ column (100 × 2.1 mm, 1.7 µm particle size) using acetonitrile and 2 mm ammonium formate in ratio 90:10 (v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode using positive electrospray ionization for indapamide and the internal standard (zolpidem tartarate). The total run time was 2.5 min. The present method was found to be linear in the concentration range of 1–50 ng/mL with the coefficient of determination 0.9987. The absolute recoveries of indapamide were 90.51–93.90%. The method was validated according the recommendations for validation of bioanalytical methods of European Medicines Agency guideline and was successfully used to analyze human whole blood samples for application in a pharmacokinetic study. Copyright © 2013 John Wiley & Sons, Ltd.     

Simple and sensitive liquid chromatographic quantitative analysis of the novel marine anticancer drug Yondelis (ET-743, trabectedin) in human plasma using column switching and tandem mass spectrometric detection

The development of a simple and sensitive assay for the quantitative analysis of the marine anticancer agent Yondelis (ET-743, trabectedin) in human plasma using liquid chromatography (LC) with column switching and tandem mass spectrometric (MS/MS) detection is described. After protein precipitation with methanol, diluted extracts were injected on to a small LC column (10 x 3.0 mm i.d.) for on-line concentration and further clean-up of the sample. Next, the analyte and deuterated internal standard were back-flushed on to an analytical column for separation and subsequent detection in an API 2000 triple-quadrupole mass spectrometer. The lower limit of quantitation was 0.05 ng mL(-1) using 100 micro l of plasma with a linear dynamic range up to 2.5 ng ml(-1). Validation of the method was performed according to the most recent FDA guidelines for bioanalytical method validation. The time needed for off-line sample preparation has been reduced 10-fold compared with an existing LC/MS/MS method for ET-743 in human plasma, employing a labor-intensive solid-phase extraction procedure for sample pretreatment. The proposed column switching method was successfully applied in phase II clinical trials with Yondelis and pharmacokinetic monitoring.     

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.     

Quantification of tizanidine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (MS/MS) method was developed and validated for the assay of tizanidine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the selected reaction monitoring mode. The assay exhibited a linear dynamic range of 50-5000 pg/mL for tizanidine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 13%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 300 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.     

A rapid and sensitive liquid chromatography/negative ion tandem mass spectrometry method for the determination of an indolocarbazole in human plasma using 96-well diatomaceous earth plates for solid-liquid extraction [correction of using internal standard (IS) 96-well diatomaceous earth plates for solid-liquid extraction

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the quantitation of a novel topoisomerase I inhibitor (indolocarbazole derivative I) in human plasma was developed to support clinical studies. Drug and internal standard were isolated from plasma by solid-liquid extraction using 96-well diatomaceous earth plates. Various extraction solvents were evaluated for extraction of I and 9% isopropyl alcohol (IPA) in methyl-tert-butyl ether (MtBE) was chosen as the optimal extraction solvent. The sensitivity of this LC/MS/MS method is 10x higher in negative ion mode using alkaline conditions than in positive ion mode using a wide range of pH's. A mobile phase with 2 mM ammonium hydroxide enhanced the sensitivity in negative ion mode over other volatile bases. The calibration curve for compound I is linear over the range 0.05-200 ng/mL in plasma and the lower limit of quantification (LLOQ) of the assay is 0.05 ng/mL, when 0.25 mL of plasma is processed. The method was fully validated and successfully applied to plasma samples from clinical studies. Performing chromatography at high pH, for enhanced negative ion sensitivity, eliminates the need for post-column addition of base. Furthermore, the 96-well diatomaceous earth plate extraction offers the following advantages over liquid-liquid extraction (LLE) or solid-phase extraction (SPE): clean sample extracts with reduced sample preparation time; increased sample throughput; no conditioning or washing steps; and a neutral eluate applicable to acid/base labile compounds.     

Determination of chlortetracycline in swine plasma by LC-ESI/MS/MS

A rapid, selective and sensitive method has been developed for the determination of chlortetracycline in swine plasma by LC-ESI/MS/MS. The method consists of a protein precipitation extraction for sample preparation and liquid chromatography ionspray tandem mass spectrometry for analysis. The plasma samples were extracted with acetonitrile and the supernatants were analyzed using an LC-ESI/MS/MS instrument. Separation was achieved using a C(8) analytical column and an isocratic mobile phase composed of 70:30 acetonitrile:0.5% formic acid in water at a flow rate of 500 microL/min. A linear (weighted 1/concentration) relationship was used to perform the calibration over an analytical range 20--2000 ppb (ng/mL). The intra-batch precision and accuracy at LLOQ, medium and high concentrations were 9.0, 11.3 and 9.9% and 97.7, 100.3 and 98.4%, respectively, and the inter-batch precision and accuracy at LLOQ, medium and high concentrations were 9.1, 8.4 and 7.4% and 95.1, 102.1 and 97.1%, respectively. This LC-ESI/MS/MS method for the determination of chlortetracycline in swine plasma has been proven to be within generally accepted criteria used for bioanalytical assay.     

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases - a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE

Bioanalytical methods using automated 96-well solid-phase extraction (SPE) and liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) are widely used in the pharmaceutical industry. SPE methods typically require manual steps of drying of the eluates and reconstituting of the analytes with a suitable injection solvent possessing elution strength weaker than the mobile phase. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well SPE by using normal-phase LC/MS/MS methods with low aqueous/high organic mobile phases, which consisted of 70-95% organic solvent, 5-30% water, and small amount of volatile acid or buffer. While the commonly used SPE elution solvents (i.e. acetonitrile and methanol) have stronger elution strength than a mobile phase on reversed-phase chromatography, they are weaker elution solvents than a mobile phase for normal-phase LC/MS/MS and therefore can be injected directly. Analytical methods for a range of polar pharmaceutical compounds, namely, omeprazole, metoprolol, fexofenadine, pseudoephedrine as well as rifampin and its metabolite 25-desacetyl-rifampin, in biological fluids, were developed and optimized based on the foregoing principles. As a result of the time saving, a batch of 96 samples could be processed in one hour. These bioanalytical LC/MS/MS methods were validated according to "Guidance for Industry - Bioanalytical Method Validation" recommended by the Food and Drug Administration (FDA) of the United States.     

A high-throughput multicomponent screening method for diuretics, masking agents, central nervous system (CNS) stimulants and opiates in human urine by UPLC-MS/MS

A simple and rapid multicomponent screening method of 130 substances for direct injections of urine samples has been developed. The fully automated method based on ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) is used for three different classes of doping agents: diuretics, central nervous system stimulants (CNS stimulants) and opiates. The samples are diluted with buffer containing internal standards (IS) by a pipetting robot system into 96-well plates. Samples are injected on a reversed phase sub 2-microm particle column connected to a fast polarity switching and rapid scanning tandem mass spectrometer with an electrospray interface. The software used to evaluate the results produced reports containing a small-sized window for each component and a data table list with flags to indicate any adverse analytical findings in the sample. The report can also be processed automatically using an application software, which interpret the data and indicate if there is a suspicious sample. One 96-well plate can be analyzed within 16 h.     

A rapid HPLC‐ESI‐MS/MS method for determination of β‐asarone,a potential anti‐epileptic agent,in plasma after oral administration of Acorus calamus extract to rats

β‐Asarone (BAS), a phenylpropanoid from Acorus calamus Linn., has shown biological effects in the management of cognitive impairment conditions such as Alzheimer's disease. The present paper describes a selective and sensitive liquid chromatography–tandem mass spectrometric method (HPLC‐MS/MS) using electrospray ionization source (ESI) for quantification of BAS in rat plasma. Briefly, the plasma samples were pre‐treated using a simple solid‐phase extraction method. The separation of BAS and the internal standard, caffeine, was achieved on an Agilent Zorbax XDB C₁₈ column (50 × 2.1 mm i.d., 5 µm) using 0.2 mL/min isocratic mobile phase flow. The detection was performed using an Applied Biosystems Hybrid Q‐Trap API 2000 mass spectrometer equipped with an ESI source operated in positive mode. Also, the developed bioanalytical method was validated as per the US FDA bioanalytical guidelines over the concentration range of 9.79–4892.50 ng/mL (r² ≥ 0.9951) for BAS from rat plasma. The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 86.92 ± 3.89, 85.30 ± 1.09 and 87.24 ± 4.03%, respectively. The applicability of the validated HPLC‐MS/MS method was demonstrated by successful measurement of BAS from plasma following oral administration of Acorus calamus rhizome extracts to three female albino Wistar rats. Copyright © 2012 John Wiley & Sons, Ltd.