Determination of polyamines in human plasma by high‐performance liquid chromatography coupled with Q‐TOF mass spectrometry

A high‐performance liquid chromatography coupled with Q‐time of flight mass spectrometry (HPLC/Q‐TOF MS) method was developed and validated for the determination of 1, 3‐diaminopropane, putrescine, cadaverine, spermidine and spermine in human plasma. The plasma samples were first pretreated by 10% HClO₄ and then derived by benzoyl chloride with 1, 6‐diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column using a gradient program. The detection was performed on a Q‐TOF MS by positive ionization mode. Calibration curve for each polyamine was obtained in the concentration range of 0.4 ~ 200.0 ng ? ml^?1, with limit of detection of 0.02 ~ 0.1 ng ? ml^?1. The intra‐ and inter‐day RSD for all polyamines were 2.5–14.0% and 2.9 ~ 13.4%, respectively. The method was applied to determine the polyamines in human plasma from cancer patients and healthy volunteers. Results showed that the mean levels of polyamines in the plasma of cancer patients were higher than that of healthy volunteers, which suggested that the plasma polyamines could be employed as cancer diagnostic indicators in clinical testing. Copyright © 2012 John Wiley & Sons, Ltd.     

Enantioselective determination of ketamine in dog plasma by chiral liquid chromatography–tandem mass spectrometry

Ketamine is an N‐methyl‐d ‐aspartate receptor antagonist that is usually used clinically as a racemic mixture. Its two enantiomers exhibit different pharmacological activities. To determine whether the enantiomers have different pharmacokinetic profiles, a chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of ketamine enantiomers in dog plasma. The enantiomers of ketamine were extracted from 50 μL of plasma by methyl tert‐butyl ether. Adequate chromatographic retention and baseline resolution of the enantiomers were achieved within a runtime of 5 min on a chiral column coated with polysaccharide derivatives, using a gradient mobile phase of acetonitrile and 10 mm ammonium bicarbonate aqueous solution. Ketamine enantiomers were detected by mass spectrometry with multiple reaction monitoring mode using the transitions of m/z 238.3 → 125.9 for the analytes and m/z 237.1 → 194.1 for carbamazepine (internal standard). The method was linear over the concentration range from 0.5 to 500 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was 0.5 ng/mL. The intra‐ and inter‐day precision was <7.3% and 8.5% for R‐ and S‐ketamine, respectively. The accuracy was 92.9–110.4% for R‐ketamine and 99.8–102.4% for S‐ketamine. The method was successfully applied to characterize the stereoselective pharmacokinetic profiles of ketamine in beagle dogs.     

Comparison of microwave‐assisted and heat reflux extraction techniques for the extraction of ten major compounds from Zibu Piyin Recipe using ultra high performance liquid chromatography with tandem mass spectrometry

Microwave‐assisted extraction and efficient ultra‐high performance liquid chromatography with triple quadrupole mass spectrometry were previously used to quickly extract and simultaneously quantify ginsenoside Rf, Ro, and Rd, 20(S)‐ginsenoside‐Rg₂, 20(R)‐ginsenoside‐Rg₂, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, lithospermic acid, and osthole from Zibu Piyin Recipe. We here showed that heat reflux extraction provides higher extraction efficiency of these target compounds but is more time consuming. Chromatographic separation was achieved on an Agilent ZORBAX RRHD Eclipse Plus C₁₈ column with a gradient mobile phase consisting of water/0.5% formic acid and acetonitrile at a flow rate of 0.2 mL/min, and detection was performed by positive and negative ion multiple‐reaction monitoring mode. All analytes showed good linearity (r, 0.9989–0.9999) within the test range, with a limit of detection of 0.002–0.180 μg/mL. The overall intra‐ and interday variations of the ten compounds were ≤2.9%, and the accuracy was evaluated using a recovery test at three concentrations and was in the range 97.61–103.18% (RSD ≤ 4.25%). The analytical results showed remarkable differences in the concentrations of the ten compounds extracted from Zibu Piyin Recipe by microwave‐assisted extraction and heat reflux extraction. These findings provide important information for determining the quality of Zibu Piyin Recipe.     

Determination of pseudo‐ginsenoside GQ in human plasma by high performance liquid chromatography–tandem mass spectrometry

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) was developed for the determination of pseudo‐ginsenoside GQ in human plasma. Liquid–liquid extraction was used to isolate the analyte from biological matrix followed by injection of the extracts onto a C₈ column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer (API‐4000 system) in multiple reaction monitoring mode using negative electrospray ionization. The mobile phase consisted of methanol–10 mm ammonium acetate (90:10, v/v) and the flow rate was 0.3 mL/min. The method was validated over the concentration range of 5.0–5000.0 ng/mL for plasma. Inter‐ and intra‐day precisions (relative standard deviation) were all within 15% and the accuracy (relative error) was ≤9.4%. The lower limit of quantitation was 5.0 ng/mL. The pseudo‐ginsenoside GQ was stable after 8 h at room temperature, 24 h at autosampler and three freeze–thaw cycles (from ?30 to 25 °C). The method was successfully applied to the pharmacokinetic study of pseudo‐ginsenoside GQ in healthy Chinese volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.     

Determination of plant growth regulators in pears by microwave‐assisted extraction and liquid chromatography with electrospray ionization mass spectrometry

A new method for the determination of six plant growth regulators, 3‐indolylacetic acid, 3‐indolepropionic acid, 2‐naphthoxyacetic acid, 2,4‐dicholrophenoxyacetic acid, 1‐naphthlcetic acid, and methyl naphthalene‐1‐acetate, in pears was established by liquid chromatography with electrospray ionization mass spectrometry. In this study, a microwave‐assisted extraction technique was first applied for the determination of plant growth regulators in fruit and three cleanup techniques were, respectively, investigated for the purification of pear samples. The chromatographic separation was performed on a Diamonsil C₁₈ column by using 0.01 mol/L formic acid/ammonium formate buffer solution (pH 3.5)/methanol (35:65, v/v) as the mobile phase with a flow rate of 0.7 mL/min in 1:1 split mode. The LODs ranged from 0.3 to 1.9 μg/kg. Under optimized conditions, the average recoveries (five replicates) for six plant growth regulators (spiked at 0.01, 0.05, and 0.5 mg/kg) ranged from 78.9 to 118.0%, and the RSDs were 1.4–10.3%.     

Free amino acids,biogenic amines,and ammonium profiling in tobacco from different geographical origins using microwave‐assisted extraction followed by ultra high performance liquid chromatography

This work describes a rapid, stable, and accurate method for determining the free amino acids, biogenic amines, and ammonium in tobacco. The target analytes were extracted with microwave‐assisted extraction and then derivatized with diethyl ethoxymethylenemalonate, followed by ultra high performance liquid chromatography analysis. The experimental design used to optimize the microwave‐assisted extraction conditions showed that the optimal extraction time was 10 min with a temperature of 60°C. The stability of aminoenone derivatives was improved by keeping the pH near 9.0, and there was no obvious degradation during the 80°C heating and room temperature storage. Under optimal conditions, this method showed good linearity (R ² > 0.999) and sensitivity (limits of detection 0.010–0.081 μg/mL). The extraction recoveries were between 88.4 and 106.5%, while the repeatability and reproducibility ranged from 0.48 to 5.12% and from 1.56 to 6.52%, respectively. The newly developed method was employed to analyze the tobacco from different geographical origins. Principal component analysis showed that four geographical origins of tobacco could be clearly distinguished and that each had their characteristic components. The proposed method also showed great potential for further investigations on nitrogen metabolism in plants.     

Determination of flomoxef in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high‐performance liquid chromatography–tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative‐ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C₁₈(2) column (3 μm , 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10–5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra‐ and inter‐day precisions were <11.8%, while the accuracy ranged from 99.6 to 109.0%. A stability study of flomoxef revealed that it could be successfully analyzed at 4ºС over 24 h, but it was unstable in solutions at room temperature during short‐term storage (4 h) and several freeze–thaw cycles. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of bisphenol A and its selected analogs in serum using pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry

Due to regulation of the use of bisphenol A, several analogs serving as bisphenol A replacements have drawn substantial attention for their adverse health effects. To investigate their occurrence in humans and identify possible pollution sources, it is necessary to develop a sensitive method for total bisphenols detection. Thus, a method based on enzymolysis and liquid‐liquid extraction followed by molecularly imprinted polymer solid‐phase extraction and pre‐column derivatization with high‐performance liquid chromatography and tandem mass spectrometry was proposed. The developed method exhibited superior selectivity and sensitivity. The matrix effect can be eliminated to a great extent. The method detection limits for eight bisphenols were 0.05～0.19 ng/mL. Satisfactory recoveries (71～119%) were obtained by spiking bovine serum at three levels (0.8, 8 and, 20 ng/mL). The method was successfully applied to determine total bisphenols in the serum samples of children. Bisphenol A, bisphenol F, bisphenol S, bisphenol B and bisphenol F were detected with concentrations from below the method detection limit to 1.65, 0.45, 0.79, 2.04 and 0.17 ng/mL, respectively. These results indicate that bisphenol A remains the major pollutant among the studied bisphenols in children, whereas threats from bisphenol A analogs should also be monitored.     

Enantioselective determination of trantinterol in rat plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry after derivatization

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of trantinterol enantiomers in rat plasma. Diphenhydramine was employed as the internal standard. The plasma samples were prepared using liquid-liquid extraction with n-hexane-dichloromethane-isopropanol (20:10:1, v/v/v) as the extractant. Trantinterol enantiomers after pre-column derivatization using diacetyl-l-tartaric anhydride (DATAAN) were separated on a C18 column using a gradient solvent programme. The mobile phase was composed of 3 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curve for each enantiomer was obtained in the concentration range of 1-80 ng/mL, with limit of quantification (LOQ) of 1 ng/mL. The intra- and inter- precision (R.S.D.) values were below 9.6% and accuracy (R.E.) was from −2.4 to 6.2% at all quality control (QC) levels. The developed method was applied to the enantioselective pharmacokinetic study of trantinterol in rats.     

In vivo metabolism study of (R)‐bambuterol in humans using ultra high performance liquid chromatography with tandem mass spectrometry

(R)‐Bambuterol, a selective β2‐adrenoceptor agonist, has been approved as a new drug for the treatment of asthma and chronic obstructive pulmonary disease by the China Food and Drug Administration and is currently under phase I clinical trials. In this study, a combined method based on ultra high performance liquid chromatography with triple quadrupole mass spectrometry and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the identification of the major metabolites of (R)‐bambuterol in human plasma and urine after an oral dose of 10 mg. The metabolites were separated by gradient elution program and different sample preparation methods were compared. Totally, 12 metabolites of (R)‐bambuterol were identified, including four metabolites in plasma and all 12 metabolites in urine. Among these, four metabolites are reported for the first time. The possible metabolic pathways of (R)‐bambuterol were subsequently proposed. The results indicated that (R)‐bambuterol was metabolized via hydrolysis, demethylation, oxygenation, glucuronidation, and sulfation pathways in vivo. This study revealed that this combined method was accurate and sensitive to identify the possible metabolites and to better understand the metabolism of (R)‐bambuterol in vivo.     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

Ultrasound/microwave‐assisted solid–liquid–solid dispersive extraction with high‐performance liquid chromatography coupled to tandem mass spectrometry for the determination of neonicotinoid insecticides in Dendrobium officinale

A one‐step ultrasound/microwave‐assisted solid–liquid–solid dispersive extraction procedure was used for the simultaneous determination of eight neonicotinoids (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiacloprid, imidaclothiz) in dried Dendrobium officinale by liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode. The samples were quickly extracted by acetonitrile and cleaned up by the mixed dispersing sorbents including primary secondary amine, C₁₈, and carbon‐GCB. Parameters that could influence the ultrasound/microwave‐assisted extraction efficiency such as microwave irradiation power, ultrasound irradiation power, temperature, and solvent were investigated. Recovery studies were performing well (70.4–113.7%) at three examined spiking levels (10, 50, and 100 μg/kg). Meanwhile, the limits of quantification for the neonicotinoids ranged from 0.87 to 1.92 μg/kg. The method showed good linearity in the concentration range of 1–100 μg/L with correlation coefficients >0.99. This quick and useful analytical method could provide a basis for monitoring neonicotinoid insecticide residues in herbs.     

Solid‐phase extraction and analysis of paroxetine in human plasma by ultra performance liquid chromatography–electrospray ionization mass spectrometry

A rapid, sensitive and rugged solid‐phase extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was developed for determination of paroxetine in human plasma. The procedure for sample preparation includes simple SPE extraction procedure coupled with Hypersil Gold C₁₈ column (100 mm ? 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow‐rate of 0.350 mL/min and fluoxetine was used as the internal standard. The analysis was performed on a triple‐quadrupole tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization. Using 500 μL plasma, the methods were validated over the concentration range 0.050–16.710 ng/mL for paroxetine, with a lower limit of quantification of 0.050 ng/mL. The intra‐ and inter‐day precision and accuracy of the quality control samples were within 10.0%. The recovery was 69.2 and 74.4% for paroxetine and fluoxetine respectively. Total run time was only 1.9 min. The method was highly reproducible and gave peaks with excellent chromatography properties. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioselective determination of acylamino acid fungicides in vegetables and fruits by chiral liquid chromatography coupled with tandem mass spectrometry

An efficient and sensitive enantioselective method for simultaneous determination of three acylamino acid fungicides in vegetables and fruits was presented by high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry. The three fungicides (benalaxyl, furalaxyl, and metalaxyl) residues in samples were extracted with acetonitrile containing 1% acetic acid and an aliquot was cleaned up with Si-(CH(2) )(3) -NH-(CH(2) )(2) -NH(2) and C(18) sorbent. Complete enantioseparation of three acylamino acid fungicides enantiomers was obtained under reversed-phase conditions on a cellulose tris (4-chloro-3-methylphenylcarbamate) column at 25°C using acetonitrile-0.1% formic acid solution (45:55, v/v) as a mobile phase. The elution orders of the eluted enantiomers were determined by a circular dichroism (CD) detector. The linearity, matrix effect, recovery, and precision were evaluated. Good linearity was obtained over the concentration range of 0.5-250 μg/L for each enantiomer in the standard solution and sample matrix calibration curves. There was no significant matrix effect for three fungicides determination based on the method. The inter-day mean recoveries, intra-day repeatability, and inter-day reproducibility varied from 81.3 to 95.7%, 2.2 to 9.4%, and 2.3 to 9.6%, respectively. The method provided high selectivity and sensitivity, and limits of quantification for enantiomers of three fungicides in vegetables and fruits were both 1 μg/kg.     

Determination of anethole trithione in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection

A rapid, sensitive and reliable high performance liquid chromatographic method coupled with tandem mass spectrometry via electrospray ionization (ESI) source (HPLC-MS/MS) has been developed and validated for the determination of anethole trithione (ATT) in human plasma. Diazepam was employed as the internal standard (IS). Sample extracts following liquid-liquid extraction were injected into the HPLC-MS/MS system. The analyte and IS were eluted isocratically on a C₁₈ column, with a mobile phase consisting of methanol and aqueous ammonium acetate solution (5 mM) (80:20, v/v) .The ions were detected by a triple quadrupole mass spectrometric detector in the positive mode. Quantification was performed using selected reaction monitoring (SRM) of the transitions m/z 240.88 → 197.91 and m/z 285.01 → 193.02 for ATT and for the IS, respectively. The analysis time for each run was 5.0 min. The calibration curve fitted well over the concentration range of 0.02-5 ng mL⁻¹, with the regression equation y = 1.1014x + 0.0003631, r = 0.9992. The intra-batch and inter-batch R.S.D.% were less than 15% at all concentration levels within the calibration range. The recoveries were more than 80%. The present method provides a modern, rapid and robust procedure for the pharmacokinetic study of ATT. Some important pharmacokinetic parameters of ATT in healthy Chinese volunteers are also given for the first time.     

A highly sensitive method for the quantification of fludrocortisone in human plasma using ultra‐high‐performance liquid chromatography tandem mass spectrometry and its pharmacokinetic application

A simple and high sensitive ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the determination of fludrocortisone in human plasma was developed and validated as per guidelines. The analyte and internal standard (IS), fludrocortisone‐d₅, were extracted from human plasma via liquid–liquid extraction using tert‐butyl methyl ether. The chromatographic separation was achieved on a Chromolith RP_18e column using a mixture of acetonitrile and 2 mm ammonium formate (70:30, v/v) as the mobile phase at a flow rate of 0.7 mL/min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The precursors to product ion transitions monitored for fludrocortisone and IS were m/z 381.2 → 343.2 and 386.2 → 348.4, respectively. The assay was validated with linear range of 40–3000 pg/mL. The intra‐ and inter‐day precisions (relative standard deviation) were within 0.49–7.13 and 0.83–5.87%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2015 John Wiley & Sons, Ltd.     

A method for rapid determination of arsenic species in vegetables using microwave‐assisted extraction followed by detection with HPLC hyphenated to inductively coupled plasma‐mass spectrometry

Driven by the significant need for characterization of the chemical speciation of arsenic in food, this work developed a method for rapid determination of four common arsenic species, namely, arsenite, arsenate, monomethyl arsenic acid, and dimethyl arsenic acid, in vegetables using microwave‐assisted extraction, followed by detection with high‐performance liquid chromatography hyphenated to inductively coupled plasma‐mass spectrometry. Initial screening results showed that microwave‐assisted extraction using 1% HNO₃ exhibited the highest overall efficiencies for all arsenic species without causing significant degradation of the organic ones. With the aid of response surface methodology, the optimum conditions established for extraction of arsenic species from vegetables were: 500 mg of freeze‐dried vegetable sample, extracted by closed vessel microwave‐assisted extraction using 10 mL of 2% v/v HNO₃ at 90°C for 17 min. Application of the method in the analysis of 24 market vegetable samples indicates that the extraction efficiencies for total arsenic species were in the range of 91.4–106%. Arsenite and arsenate were found to be the predominant arsenic species in the vegetables, which suggests that vegetable consumption could be an important route of inorganic arsenic exposure for the population with a heavy vegetable diet in arsenic polluted regions.     

Quantification of L‐ergothioneine in human plasma and erythrocytes by liquid chromatography‐tandem mass spectrometry

A sensitive analytical method has been developed and validated for the quantification of L‐ergothioneine in human plasma and erythrocytes by liquid chromatography‐tandem mass spectrometry. A commercially available isotope‐labeled L‐ergothioneine‐d₉ is used as the internal standard. A simple protein precipitation with acetonitrile is utilized for bio‐sample preparation prior to analysis. Chromatographic separation of L‐ergothioneine is conducted using gradient elution on Alltime C18 (150 mm × 2.1 mm, 5 µ). The run time is 6 min at a constant flow rate of 0.45 ml/min. The mass spectrometer is operated under a positive electrospray ionization condition with multiple reaction monitoring mode. The mass transitions of L‐ergothioneine and L‐ergothioneine‐d₉ are m/z 230 > 127 and m/z 239 > 127, respectively. Excellent linearity [coefficient of determination (r²) ≥ 0.9998] can be achieved for L‐ergothioneine quantification at the ranges of 10 to 10 000 ng/ml, with the intra‐day and inter‐day precisions at 0.9–3.9% and 1.3–5.7%, respectively, and the accuracies for all quality control samples between 94.5 and 101.0%. This validated analytical method is suitable for pharmacokinetic monitoring of L‐ergothioneine in human and erythrocytes. Based on the determination of bio‐samples from five healthy subjects, the mean concentrations of L‐ergothioneine in plasma and erythrocytes are 107.4 ± 20.5 ng/ml and 1285.0 ± 1363.0 ng/ml, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacokinetic study of dendrobine in rat plasma by ultra‐performance liquid chromatography tandem mass spectrometry

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C₁₈ (2.1 ×100 mm, 1.7 µm) column with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2–1000 ng/mL for dendrobine in rat plasma. The RSDs of intra‐day and inter‐day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright © 2015 John Wiley & Sons, Ltd.