Development of an LC‐MS/MS method for determination of bicalutamide on dried blood spots: application to pharmacokinetic study in mice

A rapid and highly sensitive assay method has been developed and validated for the estimation of bicalutamide (BCL) on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid extraction of BCL and tolbutamide (internal standard, IS) from mouse blood DBS cards using tert‐butyl methyl ether. Chromatographic separation was achieved with 5 mm ammonium acetate (pH 6.5)–acetonitrile (35:65, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 3.0 min. The MS/MS ion transitions monitored were 428.80 → 254.70 for BCL and 269.00 → 169.60 for IS. Method validation was performed as per regulatory guidelines. A linear response function was observed from 0.92 to 1911 ng/mL for BCL in mouse blood. The intra‐ and inter‐day precisions were in the ranges of 1.86–12.5 and 3.19–10.8%, respectively. This novel DBS method has been applied to a pharmacokinetic study in mice. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative analysis of enasidenib in dried blood spots of mouse blood using an increased‐sensitivity LC–MS/MS method: Application to a pharmacokinetic study

A simple, sensitive and rapid assay method has been developed and validated as per regulatory guidelines for the estimation of enasidenib on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The method employs liquid extraction of enasidenib from DBS disks of mouse whole blood followed by chromatographic separation using 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 1.0 mL/min on an Atlantis dC₁₈ column with a total run time of 2.0 min. The MS/MS ion transitions monitored were m/z 474.0 → 267.1 for enasidenib and m/z 309.2 → 251.3 for the internal standard (warfarin). The assay was linear in the range of 1.01 – 3044 ng/mL. The within‐run and between‐run precisions were in the range of 3.18 – 9.06 and 4.66 – 8.69%, respectively. Stability studies showed that enasidenib was stable on DBS cards for 1 month. This novel method has been applied to analyze the DBS samples of enasidenib obtained from a pharmacokinetic study in mice.     

A dried blood spot assay with HPLC–MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC–MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC₁₈ column using 10 mm ammonium formate–acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent–daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06–5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Exploring dried blood spot sampling technique for simultaneous quantification of antiretrovirals: lamivudine, stavudine and nevirapine in a rodent pharmacokinetic study

A high‐performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of lamivudine, stavudine and nevirapine was developed and validated in dried blood spot (DBS) cards. The analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the MRM mode using the respective [M + H]⁺ ions, m/z 230–112 for lamivudine, m/z 225–127 for stavudine, m/z 267–226 for nevirapine, m/z 383–337 for zidovudine (IS). The lower limit of quantification was 1 ng/mL for both lamivudine and stavudine and 10 ng/mL for nevirapine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The method was successfully applied to quantify them in a rat pharmacokinetic study in whole blood, plasma and DBS cards after a single oral co‐administration at the dose of 10, 2 and 13 mg/kg for lamivudine, stavudine and nevirapine, respectively, to male Wistar rats. Following oral administration the pharmacokinetic results in all the matrices are in close agreement. Thus accomplishment of this method would facilitate the ease of collection of clinical samples on DBS cards for lamivudine, stavudine and nevirapine during human clinical trials and therapeutic drug monitoring. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of dried blood spots technique for quantitative determination of topiramate using liquid chromatography–tandem mass spectrometry

An LC‐MS/MS method for determination of the anti‐epileptic drug topiramate (TPM) in dried blood spot (DBS) samples was developed and validated. DBS samples were prepared by spotting 30 μL of spiked whole blood onto FTA^TM DMPK‐C cards and drying for at least 3 h. Six‐millimetre punched spots were then extracted by using a mixture of methanol and water (90:10, v/v) with deuterated internal standard (topiramate‐d₁₂). The extracted samples were injected into a liquid chromatograph equipped with a tandem mass spectrometric detector. Negative ions were monitored in the selected reaction monitoring mode and transitions m/z 338.2 → 78.1 and m/z 350.3 → 78.1 were used for the quantitative evaluation of TPM and internal standard, respectively. The results obtained from validation were statistically evaluated according to the requirements of the European Medicines Agency and US Food and Drug Administration regulatory guidelines. The linearity of the method was checked within a concentration range from 10 to 2000 ng/mL. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC‐MS/MS method for determination of tacrolimus on dried blood spots

A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC‐ESI‐MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid–liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra‐assay, inter‐assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15–50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS‐LC‐MS/MS. Fifty samples from patients were detected by both DBS‐LC‐MS/MS and microparticle enzyme‐linked immunoassay (MEIA). TAC concentrations measured by DBS‐LC‐MS/MS method tended to be lower than those by MEIA. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid determination of rifaximin on dried blood spots by LC‐ESI‐MS

The use of blood spot collection cards is a simple way to obtain specimens for therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in a clinical setting. A high‐throughput liquid chromatography–electrospray ionization mass spectrometric (LC‐ESI‐MS) method for determination of rifaximin on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed‐phase LC on a monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI‐MS. Rifampicin was used as an internal standard (IS). The run time was within 5.0 min with a very low back‐pressure at a flow rate of 0.5 mL/min. The assay was linear from 0.1 to 10 ng/mL. The mean recovery was 98.42%. The developed method is very simple, rapid and useful for clinical applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of peptides using N‐terminal isotope coding and C‐terminal derivatization for sensitive analysis by micro liquid chromatography‐tandem mass spectrometry

Stable isotope‐coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional‐group derivatization of bioactive peptides followed by micro liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). By separating the sensitivity‐enhancement and isotope‐coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde‐d₄ to afford N‐alkylated products with different masses. This process is simple, quick and high‐yield, and accurate comparative analysis can be achieved for the mass‐differentiated peptides. Then, the carboxyl groups were derivatized with 1‐(2‐pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I–IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC‐MS/MS. In all instances, a greater than 17‐fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp‐Arg‐(Val‐d₈)‐Tyr‐Ile‐His‐Pro‐(Phe‐d₈)‐His‐Leu]. Copyright © 2016 John Wiley & Sons, Ltd.     

Study on pharmacokinetics and tissue distribution of pteryxin in mice by ultra-pressure liquid chromatography with tandem mass spectrometry

Pteryxin is a coumarin compound naturally occurring in the roots of Radix Peucedani, a commonly used as traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. An UPLC‐MS/MS method was established to quantify pteryxin in mouse plasma and tissue homogenates. Isoimperatorin was used as internal standard (IS). The method was based on protein precipitation with methanol for sample preparation. Pteryxin and IS were separated using a UPLC? BEH C₁₈ column and eluted with a mobile phase consisting of methanol and water (70:30, v/v) at a flow‐rate of 0.2 mL/min. MS/MS detection was carried out by monitoring the fragmentation of m/z 409.3–287.2 for pteryxin and m/z 271.3–185.2 for IS on a triple‐quadrupole mass spectrometer. The total run time was only 6 min. The results showed that it had good linearity over a wide concentration range (r > 0.999), and pteryxin was rapidly distributed and then eliminated from mouse plasma (t_1/2 =1.463 h). The major distribution tissues of pteryxin in mice were liver, and pteryxin was enabled to cross the blood–brain barrier owing to its low polarity. There was no long‐term accumulation of pteryxin in mouse tissues. Copyright © 2011 John Wiley & Sons, Ltd.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

Separation and identification of enzymatically prepared dephosphorylated products of myo‐inositolhexakisphosphate using LC‐MS

LC‐MS technique described here is a new way for the separation and direct determination of UV–Vis insensitive inositol phosphates (InsP₂‐InsP₆). This circumvents the need of radioisotopic labeling and post‐column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP₆ on C₁₈‐column using MeOH/H₂O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI‐MS). The LC‐MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP₆ in a sequential manner leading to InsP₂ (InsP₂·2Na, t_R 4.4–4.54 min, base peak m/z 382.9) as the end product.     

Quantitative determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood by liquid chromatography–tandem mass spectrometry with pre‐column derivation and its pharmacokinetic application

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood. DASK was pretreated using pre‐column derivatization with 2‐mercaptoethanol followed by liquid–liquid extraction with cyclohexane. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring mode via electrospray ionization source. The linear range for the determination of DASK spiked in rat whole blood (0.25 mL) was 3–3000 ng/mL. The accuracy was within 9%. Intra‐ and inter‐day precisions were no more than 16.1 and 13.3%, respectively. The validated LC‐MS/MS method was successfully applied to the preliminary pharmacokinetic study in rats. After DASK administration (60 mg/kg, p.o.) in rats, pharmacokinetic parameters were obtained, where the area under the drug concentration–time curve was 2393.7 ± 224.4 ng h/mL and the elimination half‐life was 27.6 ± 5.3 h. Copyright © 2008 John Wiley & Sons, Ltd.     

A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a UPLC‐MS/MS method for the determination of cucurbitacin B in rat plasma and application to a pharmacokinetic study

Cucurbitacin B (CuB), one of the most abundant forms of cucurbitacins, is a promising natural anticancer drug candidate. Although the anticancer activity of CuB has been well demonstrated, information regarding the pharmacokinetics is limited. A rapid, selective and sensitive UPLC‐MS/MS for CuB was developed and validated using hemslecin A (HeA) as internal standard (IS). Plasma samples were pre‐treated by liquid–liquid extraction with dichloromethane. Separation was achieved on a reversed‐phase C₁₈ column (50 × 4.6 mm, 5 µm) at 35°C using isocratic elution with water–methanol (25:75, v/v) at a flow rate of 0.3 mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with positive electrospray ionization mode. The calibration curve was linear (r > 0.995) in a concentration range of 0.3–100 ng/mL with a limit of quantification of 0.3 ng/mL. Intra‐ and inter‐day accuracy and precision were validated by percentage relative error and relative standard deviation, respectively, which were both lower than the limit of 15%. This assay was successfully applied to a pharmacokinetic study of CuB in Wistar rats. Copyright © 2015 John Wiley & Sons, Ltd.     

A UPLC–MS/MS approach for simultaneous determination of eight flavonoids in rat plasma,and its application to pharmacokinetic studies of Fu‐Zhu‐Jiang‐Tang tablet in rats

The purpose of this study is to establish and validate a UPLC–MS/MS approach to determine eight flavonoids in biological samples and apply the method to pharmacokinetic study of Fu‐Zhu‐Jiang‐Tang tablet. A Waters BEH C₁₈ UPLC column was employed with methanol/0.1% formic acid–water as mobile phases. The mass analysis was carried out in a triple quadrupole mass spectrometer using multiple reaction monitoring with negative scan mode. A one‐step protein precipitation by methanol was used to extract the analytes from blood. Eight major flavonoids were selected as markers. Our results showed that calibration curves for 3′‐hydroxypuerarin, mirificin, puerarin, 3′‐methoxypuerarin, daidzin, rutin, astragalin and daidzein displayed good linear regression (r ² > 0.9986). The intra‐day and inter‐day precisions (RSD) of the eight flavonoids at high, medium and low levels were <8.03% and the bias of the accuracies ranged from −5.20 to 6.75%.The extraction recoveries of the eight flavonoids were from 91.4 to 100.5% and the matrix effects ranged from 89.8 to 103.8%. The validated approach was successfully applied to a pharmacokinetic study in Sprague–Dawley rats after oral administration of FZJT tablet. Double peaks were emerged in curves of mean plasma concentration for 3′‐methoxypuerarin, which was reported for the first time.     

Enantioselective LC‐ESI‐MS/MS determination of dropropizine enantiomers in rat plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective and reliable LC–ESI‐MS/MS method for direct quantitation of dropropizine enantiomers namely levodropropizine (LDP) and dextrodropropizine (DDP) in rat plasma without the need for derivatization as per regulatory guidelines. Dropropizine enantiomers and carbamazepine (internal standard) were extracted from 50 μL rat plasma using ethyl acetate. LDP and DDP resolved with good baseline separation (R_s = 4.45) on a Chiralpak IG‐3 column. The mobile phase consisted of methanol with 0.05% diethylamine pumped at a flow rate of 0.5 mL/min. Detection and quantitation were done in multiple reaction monitoring mode following the transitions m/z 237 → 160 and 237 → 194 for dropropizine enantiomers and the internal standard, respectively, in the positive ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 3.23–2022 ng/mL for each enantiomer. The intra‐ and inter‐day precisions were in the ranges of 3.38–13.6 and 5.11–13.8 for LDP and 4.19–11.8 and 8.89–10.1 for DDP. Both LDP and DDP were found to be stable under different stability conditions. The method was successfully used in a stereoselective pharmacokinetic study of dropropizine enantiomers in rats following oral administration of racemate dropropizine at 100 mg/kg. The pharmacokinetic results indicate that the disposition of dropropizine enantiomers is not stereoselective and chiral inversion does not occur in rats.     

Quantitative determination of meloxicam in dog plasma by high performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method was developed to determine meloxicam in beagle dog plasma. Sample pretreatment involved a one‐step protein precipitation with methanol of 0.1 mL plasma. Analysis was performed on a Venusil ASB‐C₁₈ column with mobile phase consisting of methanol–water (containing 0.1% formic acid) (75:25, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization source. Each plasma sample was chromatographed within 4.1 min. The linear calibration curves for meloxicam was obtained in the concentration range of 10.3–4.12 × 10³ ng/mL (r ≥ 0.99). The intra‐ and inter‐day precisions (relative standard deviation) were ≤ 15%, and accuracy (relative error) was within ±7.3%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of meloxicam tablets in beagle dog.     

Determination of bencycloquidium bromide in dog plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining bencycloquidium bromide (BCQB) in beagle dog plasma. The plasma sample was deproteinized with methanol which contained l‐ethyl‐bencycloquidium bromide as internal standard, and supernantant was assayed by LC‐MS/MS. The chromatographic separation was performed on a Phenomenex C₁₈ column (100 × 2.0 mm, i.d., 3.0 μm) with a gradient programme mobile phase consisting of methanol and ammonium acetate (5 mm) containing 0.15% acetic acid and at a flow rate of 0.3 mL/min. Electrospray ionization in positive ion mode and selective reaction monitoring was used for the quantification of BCQB with a monitored transitions m/z 330.2 → 142.1 for BCQB and m/z 344.2 → 126.2 for IS. Validation results indicated that the lower limit of quantification was 0.05 ng/mL and the assay exhibited a linear range of 0.05–10.0 ng/mL and gave a correlation coefficient of 0.9998. The intra‐ and inter‐run precisions of the assay were 1.7–4.6 and 3.2–15.6%, respectively, and the intra‐ and inter‐day accuracies were ?8.8 to 1.1 and ?5.0 to 4.6%, respectively. The developed method was applied for the pharmacokinetic study of BCQB in beagle dogs following a single intranasal dose. Copyright © 2009 John Wiley & Sons, Ltd.