Determination of 3‐α‐hydroxytibolone in human plasma by LC‐MS/MS: application for a pharmacokinetic study after administration of a tibolone formulation

A new method was developed for the quantitation of 3‐α‐hydroxy tibolone, in human plasma, after oral administration of a tablet formulation containing tibolone (2.5 mg). 3‐α‐Hydroxy tibolone was extracted by a liquid–liquid procedure, using cyproterone acetate as internal standard and chlorobutane as extraction solvent. After extraction, samples were submitted to a derivatization step with p‐toluenesulfonyl isocyanate. A mobile phase consisting of acetonitrile and water (72:28 v/v) was used and chromatographic separation was achieved using Agilent XDB C₁₈ column (100 × 4.6 mm i.d.; 5 µm particle size), at 40°C. Mass spectrometric detection was performed using atmospheric pressure chemical ionization in negative mode for 3‐α‐hydroxy tibolone and in positive mode for cyproterone acetate. The fragmentation transitions were m/z 510.2 → m/z 170.1 and m/z 417.0 → m/z 357.1 for 3‐α‐hydroxy tibolone and cyproterone acetate, respectively. Calibration curves were constructed over the range 100–30,000 pg/mL and the method was shown to be specific, precise and accurate, with a mean recovery rate of 94.2% for 3‐α‐hydroxy tibolone. No matrix effect or carry‐over was detected in the samples. The validated method was applied in a pharmacokinetic study with a tibolone formulation in healthy female volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

Optimization of a validated stability‐indicating RP‐LC method for the determination of fulvestrant from polymeric based nanoparticle systems,drugs and biological samples

Fulvestrant is used for the treatment of hormone receptor‐positive metastatic breast cancer in postmenopausal women with disease progression following anti‐estrogen therapy. Several reversed‐phase columns with variable silica materials, diameters, lengths, etc., were tested for the optimization study. A good chromatographic separation was achieved using a Waters X‐Terra RP₁₈ column (250 × 4.6 mm i.d. × 5 µm) and a mobile phase, consisting of a mixture of acetonitrile–water (65:35; v/v) containing phosphoric acid (0.1%). The separation was carried out 40°C with detection at 215 nm.The calibration curves were linear over the concentration range between 1.0–300 and 1.0–200 µg/mL for standard solutions and biological media, respectively. The proposed method is accurate and reproducible. Forced degradation studies were also realized. This fully validated method allows the direct determination of fulvestrant in dosage form and biological samples. The average recovery of the added fulvestrant amount in the samples was between 98.22 and104.03%. The proposed method was also applied for the determination of fulvestrant from the polymeric‐based nanoparticle systems. No interference from using polymers and other excipients was observed in in vitro drug release studies. Therefore an incorporation efficiency of fulvestrant‐loaded nanoparticle could be determined accurately and specifically. Copyright © 2014 John Wiley & Sons, Ltd.     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of an ultra high‐performance liquid chromatographic method for the determination of a diastereomeric impurity in (+)‐pinoresinol diglucoside chemical reference substance

(+)‐Pinoresinol 4,4′‐di‐O‐β‐D ‐glucopyranoside ((+)‐PDG) is one of the major lignans with various pharmacological activities which could be isolated from Duzhong and other plant species. In this study, a diastereomeric impurity, (?)‐pinoresinol 4,4′‐di‐O‐β‐D ‐glucopyranoside ((?)‐PDG), the main impurity was identified in (+)‐PDG chemical reference substance (CRS) and a reliable chromatographic method for rapid purity determination of (+)‐PDG CRS was firstly developed. The optimal chromatographic condition was found to be using ACN/1,4‐dioxane–water (2.5:6:91.5, v/v/v) as mobile phase on a Waters Acquity UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) with column temperature of 37°C. The method was validated and applied to determine the chromatographic purity of five (+)‐PDG CRS samples. The content of (?)‐PDG in four commercial (+)‐PDG CRS was 8.47–20.30%, whereas no (?)‐PDG was detected in our in‐house prepared (+)‐PDG CRS in which purity was confirmed to be 99.80%. The above results confirmed that this method is fast and highly efficient for purity determination of the (+)‐PDG CRS.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of febuxostat in human plasma

A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with positive ion electrospray ionization was developed for the determination of febuxostat in human plasma using d₇‐febuxostat as the internal standard (IS). A simple protein precipitation was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on a Capcell PAK C₁₈ column (4.6 × 100 mm, 5 µm) using acetonitrile–5 mm ammonium acetate–formic acid (85:15:0.015, v/v/v) as the mobile phase at a flow rate of 0.6 mL/min. An Agilent 6460 electrospray tandem mass spectrometer was operated in the multiple reaction monitoring mode. The precursor‐to‐product ion transitions m/z 317 → m/z 261 (febuxsotat) and m/z 324 → m/z (261 + 262) (d₇‐febuxostat, IS) were used for quantitation. The results were linear over the studied range (10.0–5000 ng/mL), and the total analysis time for each chromatograph was 3 min. The intra‐ and inter‐day precisions were less than 7.9 and 7.2%, respectively, and the accuracy was within ±4.2%. No evidence of analyte instability in human plasma was observed storage at ?20°C for 31 days. This method was successfully applied in the determination of febuxostat concentrations in plasma samples from healthy Chinese volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4‐O‐deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 µL plasma aliquot was protein precipitated with acetonitrile–methanol (1:1, v/v) containing the internal standard vinorelbine‐d3 and 20 µL volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using isocratic elution with 1 mm ammonium acetate–ammonia buffer pH 10.5–acetonitrile–methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4‐O‐deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 µL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4‐O‐deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre‐)clinical pharmacologic studies with vinorelbine in humans and mice. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous HPLC determination of ergosterol and 22,23‐dihydroergosterol in Flammulina velutipes sterol‐loaded microemulsion

An efficient HPLC method was developed and validated for the simultaneous determination of ergosterol and 22,23‐dihydroergosterol in Flammulina velutipes sterol‐loaded microemulsions (FVSMs). The different chromatographic conditions for in vitro and in vivo determinations were investigated, with the application examined by tissue distribution. Chromatographic separation was achieved on an Inertsil ODS‐SP (250 × 4.6 mm, 5 µm) analytical column using a mobile phase of 98% methanol (in vitro), and 93% methanol for stomach samples and 96% methanol for other samples (in vivo) at 1.0 mL/min. The sterol content was detected at 282 nm. The established in vitro linearity ranges for ergosterol and 22,23‐dihydroergosterol were 0.58–72.77 µg/mL (r1 = 0.9999) and 0.59–73.25 µg/mL (r2 = 0.9999), respectively, with the biological (in vivo) samples following the same trend. The accuracy of the method was >99% (in vitro) and between 93%–108% (in vivo). The LOQ was 2.15 µg/L for ergosterol and 2.41 µg/L for 22,23‐dihydroergosterol in the in vitro studies. Also, the precisions met the acceptance criterion. These results indicate that the established HPLC method was specific, linear, accurate, precise and sensitive for the separation and simultaneous determination of ergosterol and 22,23‐dihydroergosterol. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of fenofibric acid in rat plasma

A rapid and sensitive LC‐MS/MS method for the quantification of fenofibric acid in rat plasma was developed and validated. Plasma samples were prepared by liquid–liquid extraction with a mixture of N‐hexane–dichloromethane–isopropanol (100:50:5, v/v/v). Isocratic chromatographic separation was performed on a reversed‐phase Discovery C₁₈ column (2.1 × 50 mm, 5 µm). The mobile phase was methanol–water–formic (75:25:0.25, v/v/v). Detection of fenofibric acid and the internal standard (IS) diclofenac acid was achieved by ESI MS/MS in the negative ion mode using m/z 317 → m/z 213 and m/z 294 → m/z 250 transitions, respectively. The method was linear from 0.005 to 1.250 µg/mL when 100 μL plasma was analyzed. The lower limit of quantification was 0.005 µg/mL. The intra‐ and inter‐day precision values were below 8.2%, and accuracy ranged from ?0.9 to 2.1% in all quality control samples. The recovery was 90.3–94.7% and 83.3% for fenofibric acid and IS, respectively. Total run time for each sample analysis was 2.5 min. The validated method was successfully applied to a pharmacokinetic study in six rats after oral administration of fenofibrate, the ester prodrug of fenofibric acid (equivalent to fenofibric acid 5 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid fenofibric acid determination. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of 3‐methoxypterostilbene in biological fluids by high‐performance liquid chromatography: application to pre‐clinical pharmacokinetics

A method of analysis for 3‐methoxypterostilbene [trans‐3,3′5‐trimethoxy‐4′hydroxystilbene] in biological fluids is necessary to study pharmacokinetics. A novel and simple high‐performance liquid chromatographic method was developed for the determination of 3‐methoxypterostilbene in rat serum and urine. The internal standard, pinosylvin, was added to 0.1 mL serum or urine (serum proteins were precipitated with cold acetonitrile at ?20°C). Separation was achieved with a Phenomenex® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 327 nm. The calibration curves in both matrices were linear ranging from 0.05 to 100 µg/mL, and the mean extraction efficiency was >99%. Precision of the assay for both matrices was <12% (RSD) and was within 13% for all points on the calibration curve. The limit of quantification for this method was 0.05 µg/mL. The assay was successfully applied to a preliminary study of 3‐methoxypterostilbene pharmacokinetics in a rat. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of 8‐epi PGF2α concentrations as a biomarker of oxidative stress using triple‐stage liquid chromatography/tandem mass spectrometry

F2‐isoprostanes are a family of prostaglandin F2‐like compounds that are formed by free‐radical‐catalyzed peroxidation of arachidonic acid. Several F2‐isoprostanes, but in particular 8‐epi PGF_2α, are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8‐epi PGF_2α concentrations in human plasma, whole blood, erythrocytes and urine. 8‐epi PGF_2α‐d4, a stable isotope derivative of 8‐epi PGF_2α, was used as an internal standard (IS). A 50 µL sample was focused on‐column and separated on two 3 µm particle size SUPELCOSIL? ABZ+Plus HPLC columns (15 cm × 4.6 mm and 7.5 cm × 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor‐to‐product ion transitions m/z 353.4 → 193.1 (8‐epi PGF_2α), 357.4 → 197.1 (8‐epi PGF_2α‐d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples. Copyright © 2009 John Wiley & Sons, Ltd.     

LC/MS/MS analysis of α‐tocopherol and coenzyme Q10 content in lyophilized royal jelly,beebread and drone homogenate

This study shows the results of application liquid chromatography‐tandem mass spectrometry (LC/MS/MS) for assay of the content of α‐tocopherol and coenzyme Q₁₀ in bee products of animal origin, i.e. royal jelly, beebread and drone homogenate. The biological matrix was removed using extraction with n‐hexane. It was found that drone homogenate is a rich source of coenzyme Q₁₀. It contains only 8 ± 1 µg/g of α‐tocopherol and 20 ± 2 µg/g of coenzyme Q₁₀. The contents of assayed compounds in royal jelly were 16 ± 3 and 8 ± 0.2 µg/g of α‐tocopherol and coenzyme Q₁₀, respectively. Beebread appeared to be the richest of α‐tocopherol. Its level was 80 ± 30 µg/g, while the level of coenzyme Q₁₀ was only 11.5 ± 0.3 µg/g. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantification of polygalasaponin F in rat plasma using liquid chromatography–tandem mass spectrometry and its pharmacokinetics application

A rapid and highly selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of polygalasaponin F (PF) in rat plasma was developed and validated. The chromatographic separation was achieved on a reverse‐phase Zorbax SB‐C₁₈ column (150 × 4.6 mm, 5 µm), using 2 mm ammonium acetate (pH adjusted to 6.0 with acetic acid) and acetonitrile (25:75, v/v) as a mobile phase at 30 °C. MS/MS detection was performed using an electrospray ionization operating in positive ion multiple reaction monitoring mode by monitoring the ion transitions from m/z 1091.5 → 471.2 (PF) and m/z 700.4 → 235.4 (internal standard), respectively. The calibration curve showed a good linearity in the concentration range 0.0544–13.6 µg/mL, with a limit of quantification of 0.0544 µg/mL. The intra‐ and inter‐day precisions were <9.7% in rat plasma. The method was validated as per US Food and Drug Administration guidelines and successfully applied to pharmacokinetic study of PF in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a LC‐MS/MS method for simultaneous determination of metoprolol and its metabolites, α‐hydroxymetoprolol and O‐desmethylmetoprolol,in rat plasma: application to the herb–drug interaction study of metoprolol and breviscapine

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), α‐hydroxymetoprolol (HMT) and O‐desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC‐C₁₈ column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, and post‐column splitting (1:4) was used to give optimal interface flow rates (0.2 mL/min) for MS detection; the total run time was 8.5 min. Mass spectrometric detection was achieved using a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42–7000 ng/mL for MET, 2.05‐4200 ng/mL for HMT and 1.95‐4000 ng/mL for DMT. The analytical method was successfully applied to herb–drug interaction study of MET and breviscapine after administration of breviscapine (12.5 mg/kg) and MET (40 mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

A simple,rapid and reliable liquid chromatography–mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring

A simple, rapid and reliable liquid chromatography–electrospray ionization tandem mass spectrometry method was established and validated for the determination of methotrexate in human plasma. After a straightforward protein precipitation by acetonitrile–water (70:30, v/v), methotrexate (MTX) and p‐aminoacetophenone (used as internal standard, IS) were separated on a Column C₁₈ column (50 × 2.1 mm, 3 µm; Column Technology, Fremont, CA, USA) using a gradient elution with mobile phase of acetonitrile and 0.03% acetic acid aqueous solution at a flow rate of 0.5 mL/min. The total chromatographic runtime was 5 min for each injection. Quantification detection was performed in a triple‐quadruple tandem mass spectrometer under positive mode monitoring the following mass transitions: m/z 455.3 → 308.3 for MTX and m/z 136.1 → 94.4 for IS. The calibration curve was linear over the range of 0.05–25.0 µmol/L with a lower limit of quantification of 0.05 µmol/L. The intra‐ and interday precisions were <5.2%, the accuracy varied from ?4.1 to 4.5%. The recovery was >94%. The LC‐MS/MS method showed an excellent agreement with the existing HPLC‐UV method using Passing–Bablok regression and Bland–Altman difference plot analysis. The validated LC‐MS/MS can be successfully applied to the routine therapeutic drug monitoring of MTX in clinical laboratories. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a high‐performance liquid chromatographic method for the determination of stemoninine in rat plasma after administration of Stemona tuberosa extracts

For the first time, an HPLC method was developed and validated for the determination of stemoninine in plasma after oral and intravenous administration of the extract of the roots of Stemona tuberosa to rats. Plasma samples were analyzed on a Waters reversed‐phase C₁₈ column using a gradient mobile‐phase of eluent A (water containing 0.1% formic acid and 0.2% triethylamine, pH 3.68) and eluent B (acetonitrile–water, 50:50, v/v). The flow rate was 1.0 mL/min and the detector wavelength was 210 nm. The Waters Oasis solid‐phase extraction cartridge was applied for the preparation of plasma samples with high recovery. A good linear relationship was obtained in the concentration range of 1.55–124 µg/mL (r = 0.9995). The limits of quantification and detection were 1.55 and 0.42 µg/mL, respectively. The average recoveries ranged from 91.11 to 96.43% in plasma at stemoninine concentrations of 3.10, 62.0 and 99.2 µg/mL. Intra‐ and inter‐batch coefficient of variations were 3.27–5.37% and 2.49–3.92%, respectively. This method was successfully applied to pharmacokinetic studies after oral and intravenous administration of Stemona tuberosa extract in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of a hydrophilic paclitaxel derivative, 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma by LC–MS/MS

A LC‐MS/MS method for the determination of a hydrophilic paclitaxel derivative 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma was developed to evaluate the pharmacokinetics of 7‐xylosyl‐10‐deacetylpaclitaxel in the rats. 7‐Xylosyl‐10‐deacetylpaclitaxel and docetaxel (IS for 7‐xylosyl‐10‐deacetylpaclitaxel) were extracted from rat plasma with acetic ether and analyzed on a Hypersil C₁₈ column (4.6 × 150 mm i.d., particle size 5 µm) with the mobile phase of ACN/0.05% formic acid (50:50, v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring mode. The standard curves for 7‐xylosyl‐10‐deacetylpaclitaxel in plasma were linear (r >0.999) over the concentration range of 2.0–1000 ng/mL with a weighting of 1/concentration². The method showed a satisfactory sensitivity (2.0 ng/mL using 50 µL plasma), precision (CV ≤ 10.1%), accuracy (relative error ?12.4 to 12.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma after intravenous administration of 7‐xylosyl‐10‐deacetylpaclitaxel to female Wistar rats. Copyright © 2008 John Wiley & Sons, Ltd.     

A reliable method of liquid chromatography for the quantification of acetaminophen and identification of its toxic metabolite N-acetyl-p-benzoquinoneimine for application in pediatric studies

The aim of the present study was to develop a simple, selective and reliable method to quantify acetaminophen and its toxic metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) for pediatric studies using 100 µL plasma samples, by reverse‐phase HPLC and UV detection. The assay was performed using a C₁₈ column and an isocratic elution with water–methanol–formic acid (70:30:0.15; v/v/v) as mobile phase. Linearity of the method was assayed in the range of 1–30 µg/mL for acetaminophen and 10–200 µg/mL for NAPQI, with a correlation coefficient r = 0.999 for both compounds, and inter‐ and intra‐day coefficients of variation of less than 13%. Several commonly co‐administered drugs were analyzed for selectivity and no interference with the determinations was observed. The detection and quantification limits for acetaminophen and NAPQI were 0.1 and 1 µg/mL, and 0.1 and 10 µg/mL respectively. The present method can be used to monitor acetaminophen levels using 100 µL plasma samples, which may be helpful when very small samples need to be analyzed, as in pharmacokinetics determination or drug monitoring in plasma in children. This assay is also able to detect the NAPQI for drug monitoring in patients diagnosed with acetaminophen intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative determination of sauchinone in rat plasma by liquid chromatography-mass spectrometry

A rapid, sensitive and specific method using liquid chromatography with tandem mass spectrometric detection (LC‐MS) was developed for the analysis of sauchinone in rat plasma. Di‐O‐methyltetrahydrofuriguaiacin B was used as internal standard (IS). Analytes were extracted from rat plasma by liquid–liquid extraction using ethyl acetate. A 2.1 mm i.d. × 150 mm, 5 µm, Agilent Zorbax SB‐C₁₈ column was used to perform the chromatographic analysis. The mobile phase was methanol–deionized water (80:20, v/v). The chromatographic run time was 7 min per injection and the flow‐rate was 0.2 mL/min. The tandem mass spectrometric detection mode was achieved with electrospray ionization interface in positive‐ion mode (ESI⁺). The m/z ratios [M + Na]⁺, m/z 379.4 for sauchinone and m/z 395.4 for IS were recorded simultaneously. Calibration curve were linear over the range of 0.01–5 µg/mL. The lowest limit of quantification was 0.01 µg/mL. The intra‐day and inter‐day precision and accuracy of the quality control samples were 2.94–9.42% and 95.79–108.05%, respectively. The matrix effect was 64.20–67.34% and the extraction recovery was 93.28–95.98%. This method was simple and sensitive enough to be used in pharmacokinetic research for determination of sauchinone in rat plasma. Copyright © 2011 John Wiley & Sons, Ltd.     

A simple and rapid high‐performance liquid chromatography method for determination of alendronate sodium in beagle dog plasma with application to preclinical pharmacokinetic study

A simple and rapid high performance liquid chromatographic (HPLC) method for quantifying alendronate in beagle dog plasma was developed, validated and applied to a pharmacokinetic study. The sample preparation involved coprecipitation with CaCl₂ and derivatization with o‐phthalaldehyde. Chromatographic separation was achieved on a Diamonsil? C₁₈ (250 × 4.6 mm, 5 µm) using acetonitrile–0.4% EDTA‐Na₂ (16:84, v/v) containing 0.034% of NaOH as mobile phase. The fluorimetric detector was operated at 339 nm (excitation) and 447 nm (emission). The linearity over the concentration range of 5.00–600 ng/mL for alendronate was obtained and the lower limit of quantification was 5.00 ng/mL. For each level of quality control samples, inter‐day and intra‐day precisions were less than 8.52 and 7.42% and accuracies were less than 9.07%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70 mg alendronate sodium to beagle dogs, the maximum plasma concentration (C_max) and elimination half‐life were 152 ± 27.3 and 1.75 ± 0.267 h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.