Quantification of β‐eudesmol in rat plasma using LC–MS/MS and its application to a pharmacokinetic study

A sensitive and specific LC–MS/MS assay for determination of β ‐eudesmol in rat plasma was developed and validated. After liquid–liquid extraction with ethyl ether , the analyte and IS were separated on a Capcell Pak C₁₈ column (50 × 2.0 mm, 5 μm) by isocratic elution with acetonitrile—water–formic acid (77.5:22.5:0.1, v /v/v) as the mobile phase at a flow rate of 0.4 mL/min. An ESI source was applied and operated in positive ion mode; a selected reaction monitoring scan was used for quantification by monitoring the precursor–product ion transitions of m/z 245.1 → 163.1 for β ‐eudesmol and m/z 273.4 → 81.2 for IS. Good linearity was observed in the concentration range of 3–900 ng/mL for β ‐eudesmol in rat plasma. Intra‐ and inter‐day precision and accuracy were both within ±14.3%. This method was applied for pharmacokinetic studies after intravenous bolus of 2.0 mg/kg or intragastric administration of 50 mg/kg β ‐eudesmol in rats.     

An LC–MS/MS method for quantification of demethylzeylasteral,a novel immunosuppressive agent in rat plasma and the application to a pharmacokinetic study

In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of demethylzeylasteral in rat plasma. Electrospray ionization was operated in the negative ion mode while demethylzeylasteral and oleanolic acid (internal standard) were measured by selected reaction monitoring (demethylzeylasteral: m/z 479.2 → 436.0; oleanolic acid: m/z 454.9 → 407.2). This LC–MS/MS method had good selectivity, sensitivity, accuracy and precision. The pharmacokinetic profiles of demethylzeylasteral were subsequently examined in Wistar rats after oral or intravenous administration.     

A simple LC‐MS method for determination of cyasterone in rat plasma: application to a pilot pharmacokinetic study

A simple, specific, and sensitive liquid chromatography–mass spectrometry (LC‐MS) method for determination of cyasterone in rat plasma was developed in our laboratory. Cucurbitacin B was used as an internal standard (IS). After protein precipitation with twofold volume of acetonitrile, the analyte and IS were separated on a Luna C₁₈ column (100 × 4.6 mm, i.d., 3.0 µm; Phenomenex) by isocratic elution with acetonitrile–water (80:20, v/v) as the mobile phase at a flow rate of 0.4 mL/min. An electrospray ionization source was applied and operated in the positive ion mode; selected ion monitoring scan mode was used for quantification, and the target ions m/z 543.3 for cyasterone and m/z 581.3 for IS were chosen. Good linearity was observed in the concentration range of 0.40–400 ng/mL for cyasterone in rat plasma. Intra‐day and inter‐day precision were both <7.4%. This method was proved to be suitable for pharmacokinetic studies after oral (5.0 mg/kg) or intravenous (0.5 mg/kg) administration of cyasterone in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Validated LC–MS/MS method for the quantification of sciadopitysin in rat plasma and its application to pharmacokinetic and bioavailability studies in vivo

A sensitive and rapid LC–MS/MS method was developed and validated for quantitation of sciadopitysin in rat plasma using amentoflavone as an internal standard. Sample processing was accomplished after deproteinization with 150 μL aliquot of acetonitrile. Chromatographic separation was achieved using an Agela C₁₈ column with an isocratic mobile phase comprising 2 mm ammonium acetate–acetonitrile (35:65, v/v) at a flow rate of 0.4 mL/min. Detection was performed by selection reaction monitoring on a triple‐quadrupole mass spectrometer following the transitions m/z 579 → 547 and 537 → 375 for sciadopitysin and internal standard, respectively, in the negative ionization mode. The calibration curve was linear from 2.90 to 1160 ng/mL for sciadopitysin. Intra‐ and inter‐day precisions were in the ranges 4.1–11.4 and 5.7–9.1% for sciadopitysin. Sciadopitysin was stable under different stability conditions. The validated assay was applied to pharmacokinetic and bioavailability studies in rats.     

Development of a targeted method for quantification of gypenoside XLIX in rat plasma,using SPE and LC–MS/MS

A sensitive, selective and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of gypenoside XLIX, a naturally occurring gypenoside of Gynostemma pentaphyllum in rat plasma and then validated according to the US Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation . Plasma samples were prepared by a simple solid‐phase extraction. Separation was performed on a Waters XBridgeTM BEH C₁₈ chromatography column (4.6 × 50 mm, 2.5 μm) using a mobile phase of acetonitrile and water (62.5:37.5, v /v). Gypenoside XLIX and the internal standard gypenoside A were detected in the negative ion mode using selection reaction monitoring of the transitions at m/z 1045.6 → 913.5 and 897.5 → 765.4, respectively. The calibration curve was linear (R ² > 0.990) over a concentration range of 10–7500 ng/mL with the lower quantification limit of 10 ng/mL. Intra‐ and inter‐day precision was within 8.6% and accuracy was ≤10.2%. Stability results proved that gypenoside XLIX and the IS remained stable throughout the analytical procedure. The validated LC–MS/MS method was then applied to analyze the pharmacokinetics of gypenoside XLIX after intravenous administration to rats (1.0, 2.0 and 4.0 mg/kg).     

Simultaneous determination of lamivudine,stavudine and nevirapine in human plasma by LC–MS/MS and its application to pharmacokinetic study in clinic

A new high‐throughput LC–MS/MS method for the simultaneous determination of lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma is presented, with zidovudine as an internal standard. The analytes were extracted from plasma by protein precipitation and only 150 μL plasma was needed. Chromatographic separation was achieved on a Shiseido C₈ column (150 × 2.0 mm, 5 μm) with a total run time of 6 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was developed and validated over the concentration range of 25–5000 ng/mL for 3TC and NVP and 20–4000 ng/mL for d4T. The method was validated in terms of intra‐ and inter‐day precision (≤8.6%), accuracy (within ± 8.4%), linearity and specificity. The method has been successfully applied to the pharmacokinetic study of a combination treatment of 300 mg lamivudine, 30 mg stavudine and 200 mg nevirapine in 22 healthy male volunteers under fasting conditions. Copyright © 2010 John Wiley & Sons, Ltd.     

An LC–MS/MS method for simultaneous determination of hosenkoside A and hosenkoside K from Semen Impatientis in rat plasma and its application to a pharmacokinetic study

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of two baccharane glycosides (hosenkoside A and hosenkoside K) of total saponins of Semen Impatientis in rat plasma using mogroside V as the internal standard (IS). The analytes were separated using a C₁₈ RP Agilent XDB column (1.8 μm, 50 × 2.1 mm i.d.) and detection of the compounds was done using a TSQ Quantum triple quadrupole mass spectrometer coupled with a negative electrospray ionization source under selection reaction monitoring mode. According to the US Food and Drug Administration guidelines, the established method was fully validated and the results were proved within acceptable limits. The lower limits of quantification of both analytes were 5 ng/mL. The validated method was successfully applied to a pharmacokinetic study of orally administered the total saponins of Semen Impatientis in rats.     

Validated LC–ESI–MS/MS method for the determination of tunicamycin in rat plasma: Application to a pharmacokinetic study

A liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X‐Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC–MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A–D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r²) was >0.99 for all homologs with accuracy 90.7–107.4% and precision 0.74–15.1%. The recovery of homologs was 78.6–90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench‐top for 6 h, for up to three freeze–thaw cycles, in the injector for 24 h and for 1 month at ?80 ° C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.     

Development of an LC–MS/MS method for quantification of two isomeric phenylpropenes and the application to pharmacokinetic studies in rats

Isomers β‐asarone and α‐asarone have recently been demonstrated to have differential pharmacological activities . Here, we report an LC–MS/MS method developed using acetonitrile to extract two isomeric phenylpropenes from rat plasma. Separation was achieved using a XDB‐C₁₈ column (100 × 2.1 mm; i.d., 1.8 μm) with a mobile phase of methanol–0.1% formic acid (55:45, v/v) at a flow rate of 0.3 mL/min. Calibration curves ranging from 5.20 to 2080 ng/mL for β‐asarone and from 3.68 to 1470 ng/mL for α‐asarone were linear (r² ≥ 0.9938) with the lower limits of quantification being 5.20 and 3.68 ng/mL for both isomers. Intravenous administration of β‐asarone (2.22 mg/kg) and α‐asarone (2.36 mg/kg) in rats yielded half‐lives of 13.40 ± 4.11 and 28.88 ± 7.82 min with clearance values of 0.196 ± 0.062 mL/min/kg and 0.112 ± 0.012 mL/min/kg for β‐asarone and α‐asarone, respectively.     

UPLC–MS/MS determination of flavokawain B,a novel anti‐tumor chemotherapeutic agent in rat plasma and its application to a pharmacokinetic study in rats

A sensitive, selective and rapid ultra‐performance liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of flavokawain B in rat plasma using myrislignan as an internal standard. Sample preparation was accomplished through a protein precipitation extraction process. Chromatographic resolution of flavokawain B and the IS was achieved on an Agilent XDB‐C₁₈ column (2.1 × 100 mm, 1.8 μm) using a gradient mobile phase comprising 0.1% formic acid in water and acetonitrile delivered at a flow rate of 0.5 mL/min. Flavokawain B and the IS eluted at 3.27 and 1.96 min, respectively. The total chromatographic run time was 6.0 min. A linear response function was constructed in the concentration range 0.524–1048 ng/mL. Method validation was performed as per the US Food and Drug Administration guidelines and the results met the acceptance criteria. Intra‐ and inter‐day accuracy and precision were in the ranges of ?14.3–13.2 and 3.4–11.8%, respectively. Flavokawain B was demonstrated to be stable under various stability conditions. This method has been applied to a pharmacokinetic study in rats.     

Simultaneous determination of major components of Huangqi–Honghua extract in rat plasma using LC–MS/MS and application to a pharmacokinetic study

A sensitive and reliable LC–MS/MS method was developed and validated for simultaneous quantification of the major components of Huangqi–Honghua extact in rat plasma, including hydroxysafflor yellow A (HSYA), astragaloside IV (ASIV), calycosin‐7‐O‐β‐d ‐glucoside (CAG), calycosin, calycosin‐3′‐O‐glucuronide (C‐3′‐G) and calycosin‐3′‐O‐sulfate (C‐3′‐S). After extraction by protein precipitation with acetonitrile and methanol from plasma, the analytes were separated on a Hypersil BDS C₁₈ column by gradient elution with acetonitrile and 5 mM ammonium acetate. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source switched between negative and positive modes. HSYA was monitored in negative ionization mode from 0 to 4.9 min, and ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S were determined in positive ionization mode from 4.9 to 10 min. The lower limits of quantification of the analytes were 6.25 ng/mL for HSYA, 0.781 ng/mL for CAG and 1.56 ng/mL for ASIV and calycosin. The intra‐ and inter‐assay precision (RSD) values were within 13.43%, and accuracy (RE) ranged from ?8.75 to 9.92%. The validated method was then applied to the pharmacokinetic study of HSYA, ASIV, CAG, calycosin, C‐3′‐G and C‐3′‐S in rat after an oral administration of Huangqi–Honghua extract.     

A rapid and sensitive LC–MS/MS method for analysis of iguratimod in human plasma: Application to a pharmacokinetic study in Chinese healthy volunteers

A rapid, sensitive and reproducible LC–MS/MS method was developed and validated to determine iguratimod in human plasma. Sample preparation was achieved by protein precipitation with acetonitrile. Chromatographic separation was operated on an Ultimate® XB‐C₁₈ column (2.1 × 50 mm, 3.5 μm, Welch) with a flow rate of 0.400 mL/min, using a gradient elution with acetonitrile and water which contained 2 mm ammonium acetate and 0.1% formic acid as the mobile phase. The detection was performed on a Triple Quad™ 5500 mass spectrometer coupled with an electrospray ionization interface under positive‐ion multiple reaction monitoring mode with the transition ion pairs of m/z 375.2 → 347.1 for iguratimod and m/z 244.3 → 185.0 for agomelatine (the internal standard), respectively. The method was linear over the range of 5.00–1500 ng/mL with correlation coefficients ≥0.9978. The accuracy and precision of intra‐ and inter‐day, dilution accuracy, recovery and stability of the method were all within the acceptable limits and no matrix effect or carryover was observed. As a result, the main pharmacokinetic parameters of iguratimod were as follows: C_max, 1074 ± 373 ng/mL; AUC_0–72, 13591 ± 4557 ng h/mL; AUC_0–∞, 13,712 ± 4613 ng h/mL; T_max, 3.29 ± 1.23 h; and t_1/2, 8.89 ± 1.23 h.     

Pharmacokinetics and tissue distribution of oroxin B in rats using a validated LC–MS/MS assay

A highly sensitive LC–MS/MS method was developed to measure oroxin B in rat plasma and tissue homogenates. The analyte and IS were isolated from biological matrices by a simple protein precipitation followed by centrifugation. Detection was conducted by electrospray negative‐ionization mass spectrometry in selected‐reaction monitoring mode. The assay was linear in the concentration range 4.52–904 ng/mL with intra‐ and inter‐day precision of <14.41%. It was successfully applied to the pharmacokinetics and tissue distribution studies of oroxin B after an intravenous dose of 1.0 mg/kg in rats. The results would be useful for further development of oroxin B.     

Simultaneous determination of evodiamine and its four metabolites in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography tandem mass spectrometry method was validated for simultaneous quantification of evodiamine and its metabolites 10‐hydroxyevodiamine (M1), 18‐hydroxyevodiamine (M2), 10‐hydroxyevodiamine‐glucuronide (M3) and 18‐hydroxy‐ evodiamine‐glucuronide (M4) in rat plasma for the first time. The analytes were extracted with acetonitrile and separated on a C₁₈ column within 3 min. The detection was achieved in positive selected reaction monitoring mode with precursor‐to‐product transitions at m/z 304.1 → 161.1 for evodiamine, m/z 320.1 → 134.1 for M1, m/z 320.1 → 150.1 for M2, m/z 496.2 → 134.1 for M3, m/z 496.2 → 171.1 for M4 and m/z 349.2 → 305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients >0.9991. The lower limits of quantification for evodiamine, M1, M2, M3 and M4 were 0.1, 0.1, 0.1, 0.25 and 0.25 ng mL⁻¹, respectively. Extraction recoveries and matrix effects of the analytes were within the ranges of 84.51–97.21 and 90.13–103.30%, respectively. The accuracy (relative error) ranged from −8.14 to 7.23% while the intra‐ and inter‐day precisions (relative standard deviation) were < 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study will be helpful in understanding the in vivo disposition of evodiamine.     

Development of an LC–MS/MS method for quantification of two pairs of isomeric flavonoid glycosides and other ones in rat plasma: Application to pharmacokinetic studies

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of six flavonoid glycosides – isoorientin ( 1 ), orientin ( 2 ), 2″‐O‐β ‐d ‐xylopyranosyl isoorientin ( 3 ), 2″‐O‐β ‐d ‐xylopyranosyl isovitexin ( 4 ), 6‐C‐l ‐α ‐arabipyranosyl vitexin ( 5 ) and vitexin ( 6 ) – in rat plasma using isoquercitrin as the internal standard (IS). Plasma samples were prepared by a one‐step protein precipitation with acetonitrile. Chromatographic analysis was carried out on a 25 cm C₁₈ column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. Six analytes and IS were detected through electrospray ionization in negative‐ion selection reaction monitoring mode. The mass transitions were as follows: m/z 447.2 → 327.0 for 1 , m/z 447.2 → 327.0 for 2 , m/z 579.3 → 458.9 for 3 , m/z 563.0 → 293.1 for 4 , m/z 563.0 → 353.0 for 5 , m/z 431.1 → 311.1 for 6 , and m/z 463.1 → 300.2 for IS. Calibration curves exhibited good linearity (r ² > 0.9908) over a wide concentration range for all compounds. Intra‐ and inter‐day precision (RSD, %) at four different levels were both <14.2% and the accuracy (RE, %) ranged from −11.9 to 12.0%. The extraction recoveries of the six components ranged from 88.2 to 103.6%. The validated assay was successfully applied to the pharmacokinetic studies of the six components in male rat plasma after intravenous administration of total flavonoids of Scorzonera austriaca Wild.     

Quantification of metronidazole in human plasma using a highly sensitive and rugged LC–MS/MS method for a bioequivalence study

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography–tandem mass spectrometry using metronidazole‐d4 as the internal standard (IS). The analyte and the IS were extracted from 100 μL plasma by liquid–liquid extraction. The clear samples obtained were chromatographed on an ACE C₁₈ (100 × 4.6 mm, 5 μm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 μg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.     

Development and validation of an LC–MS/MS method for the determination of hinokiflavone in rat plasma and its application to a pharmacokinetic study

Hinokiflavone has drawn a lot of attention for its multiple biological activities. In this study, a sensitive and selective method for determination of hinokiflavone in rat plasma was developed for the first time, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Amentoflavone was used as an internal standard. Separation was achieved on a Hypersil Gold C₁₈ column with isocratic elution using methanol–water (65:35, v /v) as mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the negative electrospray mode with selected reaction monitoring was used to detect the transitions of m/z 537 → 284 for hinokiflavone and m/z 537 → 375 for IS. The LOQ was 0.9 ng/mL with a linear range of 0.9–1000 ng/mL. The intra‐ and inter‐day accuracy (RE%) ranged from −3.75 to 6.91% and from −9.20 to 2.51% and the intra‐ and inter‐day precision (RSD) was between 0.32–14.11 and 2.85–10.04%. The validated assay was successfully applied to a pharmacokinetic study of hinokiflavone in rats. The half‐life of drug elimination at the terminal phase was 6.10 ± 1.86 h, and the area under the plasma concentration‐time curve from time zero to the time of last measurable concentration and to infinity values obtained were 2394.42 ± 466.86 and 2541.93 ± 529.85 h ng/mL, respectively.     

A UPLC–MS/MS method for comparative pharmacokinetics study of morusin and morin in normal and diabetic rats

The aim of this study was to establish and validate a rapid, selective and reliable ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for simultaneous quantitations of morin and morusin, and to investigate their pharmacokinetics difference between normal and diabetic rats after oral administration. Plasma samples were pretreated via protein precipitation with acetonitrile. Genkwanin was used as internal standard (IS). Analytes and IS were separated on a Thermo Hypersil Gold C₁₈ column (50 × 4.6 mm, 3 μm) using gradient elution. The mobile phase consisted of acetonitrile and 0.1% formic acid in water at a flow rate of 0.5 mL/min. Mass spectrometry detection was carried out by means of negative electrospray ionization source and multipe‐reaction monitoring mode. The transitions of m/z 300.9 → 151.2 for morin, m/z 419.2 → 297.1 for morusin and m/z 283.1 → 268.2 for IS were chosen for quantification. Calibration curves were linear in the range of 1.01–504.2 ng/mL (r² ≥ 0.99) for morin and 1.02–522.3 ng/mL (r² ≥ 0.99) for morusin. The lower limit of quantification was 1.02 ng/mL for morin and 1.05 ng/mL for morusin. The extraction recovery was >85.1% for each analyte. No obvious matrix effect was observed under the present UPLC–MS/MS conditions during all of the bioanalysis. The stability study demonstrated that morin and morusin remained stable during the whole analytical procedure. The method was successfully applied to support the pharmacokinetic comparisons of morin and morusin between normal and diabetic rats.     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A single LC–MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion‐pair solid‐phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed‐phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile–15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]⁺/60.1 for metformin and m/z 467.1 [M + CH₃COO]^?/329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.