Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development,validation and application to a pharmacokinetic study of curcumin liposome

This paper describes a sensitive, specific and rapid high‐performance liquid chromatography (HPLC) method for the determination of curcumin in rat plasma. After a simple step of protein precipitation in 96‐well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse‐phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C₁₈ analytical column (4.6 × 100 mm, 5 µm) using acetonitrile–5% acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with good linearity (r² ≥ 0.999) over the linear range 1–500 ng/mL. All the validation data, such as accuracy and precision, were within the required limits. A run time of 3.0 min for each sample made high‐throughput bioanalysis possible. The assay method was successfully applied to the study of the pharmacokinetics of curcumin liposome in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and optimization of a supercritical fluid chromatography tandem mass spectrometry method for the high‐throughput determination of nimodipine in beagle plasma

A simple, sensitive, and efficient supercritical fluid chromatography with tandem mass spectrometry method was established for the determination of nimodipine in beagle plasma. One‐step protein precipitation with acetone was used to extract the analytes from the plasma. Nitrendipine was used as the internal standard. The chromatographic separation was achieved on an ACQUITY UPC²? BEH 2‐EP column, and a gradient elution program was applied at a flow rate of 1.5 mL/min. The detection was carried out on a triple quadrupole tandem mass spectrometer with an electrospray ionization source operating in positive ion mode. Quantification was performed using multiple reaction monitoring of the transitions of m/z 419.3→301.3 for nimodipine and m/z 361.4→315.2 for nitrendipine. A satisfactory linearity was obtained over the concentration range of 0.5–800 ng/mL (r > 0.996). The intra‐ and interday precision and accuracy results were <9.1% across the quality control levels. The peak concentration and area under concentration‐time curve (0–720 min) values of the test and reference formulations were 279.28 ± 211.46 and 265.13 ± 149.26 ng/mL, 25608.00 ± 17553.65 and 28553.67 ± 20207.92 ng·min/mL, respectively. The validated method was successfully applied to reveal the pharmacokinetic profiles of nimodipine in beagle dogs after oral administration. Moreover, the analytical method could be used for further bioequivalence studies.     

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE‐based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4‐dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, ε‐aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs–Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.     

Rapid and sensitive LC‐MS method for pharmacokinetic study of vinorelbine in rats

A rapid and sensitive LC‐electrospray ionization‐MS method was developed for determining vinorelbine in rat plasma. A 100 µL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS‐3 C₁₈ (2.1 × 50 mm, 5 µm) column. The selected ion monitoring ions [M + H]⁺ were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1–1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

High‐throughput LC–MS/MS method with 96‐well plate precipitation for the determination of arotinolol and amlodipine in a small volume of rat plasma: Application to a pharmacokinetic interaction study

A rapid, sensitive, and selective liquid chromatography with tandem mass spectrometry method was developed and fully validated for the simultaneous quantification of arotinolol and amlodipine in rat plasma. Two internal standards were introduced with metoprolol as the internal standard of arotinolol and (S)‐amlodipine‐d4 as the internal standard of amlodipine. The analytes were isolated from 50.0 μL plasma samples by a simple protein precipitation using acetonitrile. The chromatographic separation was achieved in 5 min on a C18 column. The mobile phase consisted of phase A 5% methanol and phase B 95% methanol (both containing 0.5% formic acid and 5 mM ammonium acetate) and was delivered in gradient elution at 0.300 mL/min. Quantification was performed in multiple reaction monitoring mode with the transition m/z 372.1 → 316.1 for arotinolol, m/z 268.2 → 116.2 for metoprolol, m/z 409.1 → 238.1 for amlodipine and m/z 413.1 → 238.1 for (S)‐amlodipine‐d4. Linearity was obtained over the range of 0.200–40.0 ng/mL for arotinolol (r² = 0.9988) and 0.500–100 ng/mL for amlodipine (r² = 0.9985) in rat plasma. The validated data have met the acceptance criteria in FDA guideline. This method was successfully applied to a pharmacokinetic interaction study in rats, and the results indicated that there was no significant drug–drug interaction between arotinolol and amlodipine.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Hydrophilic interaction chromatography‐tandem mass spectrometric analysis of irbesartan in human plasma: Application to pharmacokinetic study of irbesartan

A hydrophilic interaction chromatography‐tandem mass spectrometric method (HILIC/MS/MS) for the determination of irbesartan in human plasma was developed. Irbesartan and losartan (internal standard) were extracted from human plasma with ethyl acetate at acidic pH. The analytes were analyzed on a Luna HILIC column with the mobile phase of ACN–ammonium formate (50 mM, pH 6.5) (96:4, v/v) and detected by ESI MS/MS in the selected reaction monitoring mode. The standard curve was linear (r² = 0.9981) over the concentration range of 10–2500 ng/mL and the lower LOQ was 10 ng/mL using 100 μL of plasma sample. The CV and relative error for intra‐ and interassay at four QC levels were 2.9 to 8.1% and –2.7 to 2.3%, respectively. There were less absolute and relative matrix effects for irbesartan and losartan. The present method was successfully applied to the pharmacokinetic study of irbesartan after oral dose of irbesartan (150 mg tablet) to male healthy volunteers.     

Development of a method for the determination of vardenafil in human plasma by high performance liquid chromatography with UV detection

A simple high‐performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed‐phase Kromasil KR 100 C₁₈ (5 µm particle size) column using a mobile phase of acetonitrile–potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10–1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Simple and sensitive LC‐ESI‐MS method for the quantitation of sildenafil in plasma samples

Sildenafil is a selective inhibitor of phosphodiesterase type 5 enzyme. A simple and sensitive LC‐MS assay was developed to determine the concentration of sildenafil in human plasma. Sildenafil and omeprazole (internal standard) were extracted from the plasma with diethyl ether. The extract was evaporated under nitrogen and the residue was constituted with ACN and injected onto Novapak C₁₈ column (75×3.9 mm, 4 μm). The mobile phase consisted of 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid and was delivered isocratically at a flow rate of 0.2 mL/min. Sildenafil and omeprazole were monitored using a positive electrospray mode with single‐ion recording set at m/z 475 and 346, respectively, which are consistent with [M+H]⁺ molecular ions, and the run time was less than 5 min. The detection limit of sildenafil was 0.5 ng/mL, and the calibration curve was linear between 0.5 and 2000 ng/mL (R²>0.99). Within‐ and between‐day coefficients of variation were less than 7%. This method has been successfully used to measure sildenafil plasma concentrations in a beagle dog model following an oral administration of the drug.     

Development and validation of a rapid and sensitive assay for the determination of anisodamine in 50 μL of beagle dog plasma by LC–MS/MS

A simple, rapid, high‐throughput, and highly sensitive LC–MS/MS was developed to determine anisodamine in a small volume (50 μL) of beagle dog plasma using atropine sulfate as the internal standard. The analyte and internal standard were isolated from 50 μL plasma samples after a one‐step protein precipitation using Sirocco 96‐well protein precipitation filtration plates. The separation was accomplished on a Hanbon Hedera CN column (100 × 4.6 mm, 5 μm) and the run time was 4 min. A Micromass Quatro Ultima mass spectrometer equipped with an ESI source was operated in the multiple reaction monitoring mode with the precursor‐to‐product ion transitions m/z 306.0→140.0 (anisodamine) and 290.0→123.9 (atropine) used for quantitation. The method was sensitive with a low LOQ of 0.05 ng/mL, and good linearity in the range 0.05–50 ng/mL for anisodamine (r² ≥ 0.995). All the validation data, such as accuracy, intra‐ and interrun precision, were within the required limits. The method was successfully applied to the pharmacokinetic study of anisodamine hydrochloride injection in beagle dogs.     

Quantitative determination of Phakellistatin 13, a new cyclic heptapeptide,in rat plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, followed by a 96-well protein precipitation, has been developed and fully validated for the determination of Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated from the sponge Phakellia fusca Thiele, in rat plasma. After protein precipitation of the plasma samples (50 μL) in a 96-well plate by methanol (200 μL) containing the internal standard Pseudostellarin B (20 ng/mL), the plate was vortex mixed for 3 min. Following filtration for 5 min, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on an XB-C18 analytical column (5 μm, 50 mm × 4.6 mm i.d.) using an eluent of methanol–water (85:15, v/v) and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatographic run time of 5.0 min. The method was sensitive with a lower limit of quantification (LLOQ) of 0.1 ng/mL, with good linearity (r > 0.999) over the quantitation range of 0.1–5 ng/mL. The validation results demonstrated that this method was significantly specific, accurate, precise, and was successfully applied in measuring levels of PK13 in rat plasma following intravenous administration of 20, 50, and 100 μg/kg of peptide in rats, respectively, which was suitable for the preclinical pharmacokinetic studies on PK13.     

Simultaneous quantification of imatinib and its main metabolite N‐demethyl‐imatinib in human plasma by liquid chromatography–tandem mass spectrometry and its application to therapeutic drug monitoring in patients with gastrointestinal stromal tumor

The aim of this study was to improve and validate a more stable and less time‐consuming method based on liquid chromatography and tandem mass spectrometry (LC‐ MS/MS) for the quantitative measurement of imatinib and its metabolite N‐ demethyl‐imatinib (NDI) in human plasma. Separation of analytes was performed on a Waters XTerra RP₁₈ column (50 × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of methanol–acetonitrile–water (65:20:15, v /v/v) with 0.05% formic acid at a flow‐rate of 0.2 mL/min. The Quattro MicroTM triple quadruple mass spectrometer was operated in the multiple‐reaction‐monitoring mode via positive electrospray ionization interface using the transitions m /z 494.0 → 394.0 for imatinib, m /z 479.6 → 394.0 for NDI and m /z 488.2 → 394.0 for IS. The method was linear over 0.01–10 μg/mL for imatinib and NDI. The intra‐ and inter‐day precisions were all <15% in terms of relative standard deviation, and the accuracy was within ±15% in terms of relative error for both imatinib and NDI. The lower limit of quantification was identifiable and reproducible at 10 ng/mL. The method was sensitive, specific and less time‐consuming and it was successfully applied in gastrointestinal stromal tumor patients treated with imatinib.     

Assay of free captopril in human plasma as monobromobimane derivative,using RPLC/(+)ESI/MS/MS: validation aspects and bioequivalence evaluation

A sensitive method for determination of free captopril as monobromobimane derivative in plasma samples is discussed. The internal standard (IS) was 5‐methoxy‐1H‐benzimidazole‐2‐thiol. Derivatization with monobromobimane immediately after blood collection and plasma preparation prevents oxidation of captopril to the corresponding disulfide compound and enhances the ionization yield. Consequently, derivatization enhances sample stability and detection sensitivity. Addition of the internal standard was made immediately after plasma preparation. The internal standard was also derivatized by monobromobimane, as it contains a thiol functional group. Preparation of plasma samples containing captopril and IS derivatives was based upon protein precipitation through addition of acetonitrile, in a volumetric ratio 1:2. The reversed‐phase liquid chromatographic separation was achieved on a rapid resolution cartridge Zorbax SB‐C₁₈, monitored through positive electrospray ionization and tandem MS detection using the multiple‐reaction monitoring mode. Transitions were 408–362 amu for the captopril derivative and 371–260 amu for the internal standard derivative. The kinetics of captopril oxidation to the corresponding disulfide compound in plasma matrix was also studied using the proposed method. A linear log–log calibration was obtained over the concentration interval 2.5–750 ng/mL. A low limit of quantitation in the 2.5 ng/mL range was obtained. The analytical method was fully validated and successfully applied in a three‐way, three‐period, single‐dose (50 mg), block‐randomized bioequivalence study for two pharmaceutical formulations (captopril LPH 25 and 50 mg) against the comparator Capoten 50 mg. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of pterostilbene in rat plasma by a simple HPLC‐UV method and its application in pre‐clinical pharmacokinetic study

A simple HPLC‐UV method was developed and validated for the quantification of pterostilbene (3,5‐dimethoxy‐4'‐hydroxy‐trans‐stilbene), a pharmacologically active phytoalexin in rat plasma. The assay was carried out by measuring the UV absorbance at 320 nm. Pterostilbene and the internal standard, 3,5,4'‐trimethoxy‐trans‐stilbene eluted at 5.7 and 9.2 min, respectively. The calibration curve (20–2000 ng/mL) was linear (R² > 0.997). The lower limits of detection and of quantification were 6.7 and 20 ng/mL, respectively. The intra‐ and inter‐day precisions in terms of RSD were all lower than 6%. The analytical recovery ranged from 95.5 ± 3.7 to 103.2 ± 0.7% while the absolute recovery ranged from 101.9 ± 1.1 to 104.9 ± 4.4%. This simple HPLC method was subsequently applied in a pharmacokinetic study carried out in Sprague–Dawley rats. The terminal elimination half‐life and clearance of pterostilbene were 96.6 ± 23.7 min and 37.0 ± 2.5 mL/min/kg, respectively, while its absolute oral bioavailability was 12.5 ± 4.7%. Pterostilbene appeared to have better pharmacokinetic characteristics than its natural occurring analog, resveratrol. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of acyclovir,ganciclovir, and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine in human plasma using high‐performance liquid chromatography

Acyclovir, ganciclovir and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine are active in vitro against the Epstein–Barr virus (EBV) but their in vivo anti‐EBV activity is not well understood. We developed a novel, sensitive high‐performance liquid chromatography assay with ultraviolet detection for measuring acyclovir, ganciclovir and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine in human plasma to identify quantitative relationships between in vitro anti‐EBV activity and therapeutic response. Characteristics of the assay include a low plasma volume (200 µL), perchloric acid protein precipitation, use of penciclovir as the internal standard, run times less than 8 min and a 50 ng/mL lower limit of quantification. The within‐ and between‐assay variability is 0.7–4.8 and 1.0–7.9%, respectively. Accuracy for all three drugs ranges from 89.5 to 106.4% for four quality controls (50, 100, 1000 and 10,000 ng/mL). This assay supports pharmacokinetic and pharmacodynamic studies of candidate anti‐EBV drugs in children and adults with EBV infections. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid and sensitive determination of acetylsalicylic acid and salicylic acid in plasma using liquid chromatography–tandem mass spectrometry: application to pharmacokinetic study

A simple and sensitive analytical method using liquid chromatography–tandem mass spectrometry (LC/MS/MS) for determination of acetylsalicylic acid (aspirin, ASA) and its major metabolite, salicylic acid (SA), in animal plasma has been developed and validated. Both ASA and SA in plasma samples containing potassium fluoride were extracted using acetonitrile (protein precipitation) with 0.1% formic acid in it. 6‐Methoxysalicylic acid was used as the internal standard (IS). The compounds were separated on a reversed‐phase column. The multiple reaction monitoring mode was used with ion transitions of m/z 178.9 → 136.8, 137.0 → 93.0 and 167.0 → 123.0 for ASA, SA and IS, respectively. The lower limits of quantification for ASA and SA were 3 and 30 ng/mL, respectively. The developed method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after p.o. and i.v. administration of 1 mg/kg to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive and robust LC‐ESI‐MS/MS method for simultaneous quantitation of simvastatin acid,amlodipine and valsartan in human plasma: application to a clinical pharmacokinetic study

A high‐throughput, simple, highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of simvastatin acid (SA), amlodipine (AD) and valsartan (VS) with 500 µL of human plasma using deuterated simvastatin acid as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using electrospray ionization. The assay procedure involved precipitation of SA, AD, VS and IS from plasma with acetonitrile. The total run time was 2.8 min and the elution of SA, AD, VS and IS occurred at 1.81, 1.12, 1.14 and 1.81 min, respectively; this was achieved with a mobile phase consisting of 0.02 m ammonium formate (pH 4.5):acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X‐Terra C₁₈ column. A linear response function was established for the range of concentrations 0.5–50 ng/mL (r > 0.994) for VS and 0.2–50 ng/mL (r > 0.996) for SA and AD. The method validation parameters for all three analytes met the acceptance as per FDA guidelines. This novel method has been applied to human pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous quantification and rat pharmacokinetics of formononetin‐7‐O‐β‐d‐glucoside and its metabolite formononetin by high‐performance liquid chromatography–tandem mass spectrometry

Formononetin‐7‐O‐β‐d ‐glucoside has been proved to have significant anti‐inflammatory effect. To evaluate its rat pharmacokinetics, a rapid, sensitive, and specific liquid chromatography–tandem mass spectrometry method has been developed and validated for the quantification of formononetin‐7‐O‐β‐d ‐glucoside and its main metabolite formononetin in rat plasma. Samples were pretreated using a simple protein precipitation and the chromatographic separation was performed on a C₁₈ column by a gradient elution using a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Both analytes were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. The assay showed wide linear dynamic ranges of both 0.10–100 ng/mL, with acceptable intra‐ and inter‐batch accuracy and precision. The lower limits of quantification were both 0.10 ng/mL using 50 μL of rat plasma for two analytes. The method has been successfully used to investigate the oral pharmacokinetic profiles of both analytes in rats. After oral administration of formononetin‐7‐O‐β‐d ‐glucoside at the dose of 50 mg/kg, it was rapidly absorbed in vivo and metabolized to its metabolite formononetin. The plasma concentration‐time profiles both showed double‐peak phenomena, which would be attributed to the strong enterohepatic circulation of formononetin‐7‐O‐β‐d ‐glucoside.     

Simultaneous determination of ferulic acid,paeoniflorin, and albiflorin in rat plasma by ultra‐high performance liquid chromatography with tandem mass spectrometry: Application to a pharmacokinetic study of Danggui‐Shaoyao‐San

A rapid, selective, and sensitive ultra‐high performance liquid chromatography‐tandem mass spectrometry method was developed for simultaneous determination of ferulic acid, paeoniflorin, and albiflorin, the major active constituents of Danggui‐Shaoyao‐San, in rat plasma using geniposide as the internal standard. The plasma samples were processed by protein precipitation with acetonitrile, and then separated on a Shim‐Pack XR‐ODS C₁₈ column (75 mm × 3.0 mm, 2.2 μm) using gradient elution program with a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.4 mL/min. The detection was achieved on a 3200 QTRAP mass spectrometer equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reaction monitoring mode by monitoring the fragmentation of m/z 192.9→134.0 for ferulic acid, m/z 525.0→120.9 for paeoniflorin, m/z 525.2→121.0 for albiflorin, and m/z 433.1→225.1 for the internal standard, respectively. The calibration curve was linear in the range of 5–2500 ng/mL for all the three analytes (r ≥ 0.9972) with the lower limit of quantitation of 5 ng/mL. The intraday and interday precisions were below 12.1% for all the analytes in terms of relative standard deviation, and the accuracy was within ±11.5% in terms of relative error. The extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of ferulic acid, paeoniflorin, and albiflorin after oral administration of Danggui‐Shaoyao‐San to rats.