In vivo microdialysis with ultra performance liquid chromatography–mass spectrometry for analysis of tetramethylpyrazine and its interaction with borneol in rat brain and blood

Tetramethylpyrazine (TMP) has been widely used in the treatment of ischemic cerebrovascular disease. However, the mechanism of TMP and how to increase its bioavailability need to be further explored. In our study, an in vivo microdialysis sampling technique coupled with ultra‐performance liquid chromatography–mass spectrometry method was developed to investigate the pharmacokinetic properties of TMP and its interaction with different doses of borneol (BO) in rats. Linearity of TMP in brain and blood dialysates exhibited good linear relationships over the concentration range of 0.991–555.14 ng/mL. The specificity, linearity, accuracy, precision, matrix effect and stability were within acceptable ranges. The results demonstrated that BO had a marked impact on the pharmacokinetic properties of TMP. After co‐administration, the areas under the concentration–time curve (AUC) of TMP in brain and blood were significantly increased. Meanwhile, the peak concentration of TMP in brain was also enhanced. The AUC_Brain/AUC_Blood of TMP, increased from 44% to 56 and 60.8% after co‐administration with BO (15 and 30 mg/kg). The pharmacodynamic results showed that TMP co‐administration with BO enhanced the cerebral blood flow during the period of ischemia and reduced the infarct volume. Overall, it might be an effective way to treat stroke to use TMP co‐administered with BO.     

Assessment of pharmacokinetics,bioavailability and protein binding of anacetrapib in rats by a simple high‐performance liquid chromatography–tandem mass spectrometry method

Anacetrapib is a potent and selective CETP inhibitor and is undergoing phase III clinical trials for the treatment of dyslipidemia. A simple and sensitive high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the quantification of anacetrapib in rat plasma was developed and validated using an easily purchasable compound, chlorpropamide, as an internal standard (IS). A minimal volume of rat plasma sample (20 μL) was prepared by a single‐step deproteinization procedure with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed using selected reaction monitoring modes at the mass/charge transitions m/z 638 → 283 for anacetrapib and m/z 277 → 175 for IS. The assay was validated to demonstrate the selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. The lower limit of quantification was 5 ng/mL. This LC‐MS/MS assay was successfully applied in the rat plasma protein binding and pharmacokinetic studies of anacetrapib. The fraction of unbound anacetrapib was determined to be low (ranging from 5.66 to 12.3%), and the absolute oral bioavailability of anacetrapib was 32.7%.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative in vivo pharmacokinetics study of affeic acid,isoferulic acid and ferulic acid in crude and three different prepared Cimicifuga foetida L.

Our study investigated the differences in pharmacokinetics of three major components of crude Cimicifuga foetida L. and its fried product and honey- and liquor-prepared products. A rapid and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry approach was established for determing caffeic acid, isoferulic acid and ferulic acid in rat plasma. The approach has good linearity, precision, accuracy, recovery and stability. Phenolic acid was rapidly absorbed. The times to peak concentration were shorter in the processed group than those for the crude product, with their values of <30 min. The peak concentration values of caffeic acid and isoferulic acid were higher in the crude group than in the processed groups (p < 0.05). Area under the curve values of the three phenolics in the crude group were significantly higher than those of the processed groups (p < 0.05).     

A validated surrogate analyte UPLC–MS/MS assay for quantitation of TUDCA,TCDCA, UDCA and CDCA in rat plasma: Application in a pharmacokinetic study of cultured bear bile powder

Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become necessary because of protection measures for this endangered species. The ingredients of in vitro cultured bear bile powder (CBBP) include tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA, and it has pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, the pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC–MS method using stable isotopes as surrogate analytes (D4-TUDCA, D4-TCDCA, D4-UDCA and D4-CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975–0.9994), precision (RSD intra-day, 0.72–9.35%; inter-day, 3.82–9.02%), accuracy (RE, −12.42–5.67%) and matrix effect (95.53–99.80%), along with analyte recovery (95.90–98.82%) and stability (89.48–101.81%) of surrogate analytes, and precision (RSD intra-day, 1.06– 11.51%; inter-day, 2.23– 11.38%), accuracy (RE, −7.40–10.76%) and stability (87.37–111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed the critical in vivo properties of both bear bile preparations.     

Accurate determination of a novel vasodilatory β‐blocker TJ0711 using LC‐MS/MS: Resolution of an isobaric metabolite interference in dog plasma

A rapid, robust and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for bioanalysis of TJ0711, a novel vasodilatory β‐blocker in dog plasma. This assay is able to chromatographically separate TJ0711 from its isobaric metabolite as well as glucuronide conjugates. Chromatographic separation was achieved on a Welch Ultimate‐XB C₁₈ column (2.1 × 100 mm, 3 μm). The analyte and internal standard (propranolol) were extracted from plasma by liquid–liquid extraction using ethyl acetate. The mass spectrometric detection was carried out in positive ion multiple reaction monitoring mode. Good linearity was obtained over the concentration range of 0.5–500 ng/mL (r > 0.99) for TJ0711. Moreover, the method had good accuracy (RE ranging from −2.70 to −0.32%) and precision (RSD < 7.55%). TJ0711 was stable in dog plasma for at least 6 h at ambient temperature, for at least 30 days at −20°C and after three freeze–thaw cycles. This method was successfully applied to a preclinical pharmacokinetic study and the results demonstrated linear pharmacokinetics of TJ0711 over a dose range from 0.03 to 0.3 mg/kg. No significant gender differences were observed in TJ0711 plasma pharmacokinetic parameters.     

Development and validation of an HPLC method for quantitation of BA‐TPQ,a novel iminoquinone anticancer agent,and an initial pharmacokinetic study in mice

We herein describe the development and validation of an HPLC method for the quantitation of 7‐(benzylamino)‐1,3,4,8‐tetrahydropyrrolo [4,3,2‐de]quinolin‐8(1H)‐one (BA‐TPQ), a newly synthesized iminoquinone anticancer agent. BA‐TPQ was extracted from plasma and tissue samples by first precipitating proteins with acetonitrile followed by a liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out using a gradient flow rate on a Zorbax SB C₁₈ column, and the effluent was monitored by UV detection at 346 nm. The method was found to be precise, accurate, and specific, with a linear range of 3.91–1955.0 ng/mL in plasma, 19.55–1955.0 ng/mL in spleen, brain, and liver homogenates and 19.55–3910.0 ng/mL in heart, lung and kidney homogenates. The method was stable under all relevant conditions. Using this method, we also carried out an initial study determining plasma pharmacokinetics and tissue distribution of BA‐TPQ in mice following intravenous administration. In summary, this simple and sensitive HPLC method can be used in future preclinical and clinical studies of BA‐TPQ.     

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.     

A new rapid ultra‐performance liquid chromatography method for the pharmacokinetic and bioavailability study of diclofenac sodium aqueous injection and lipid microsphere injection in rats

A novel, rapid and selective ultra performance liquid chromatography mass spectrometric method had been developed for the pharmacokinetic study of diclofenac sodium (DS) after single intravenous injection of DS aqueous injection and DS lipid microsphere (LM) injection in rats. Ketoprofen (KP) was used as internal standard. Samples were treated by a one‐step liquid liquid extraction. Separation was performed on an Acquity UPLC? BEH C₁₈ column (50 × 2.1 mm i.d., 1.7 μm). The mobile phase consisted of acetonitrile–0.1% ammonium hydroxide aqueous solution (20 : 80, v/v) initially in the gradient mode. The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with multiple‐reaction monitoring mode. Standard curves showed good linearity (r > 0.99) from the plasma concentration of 0.1–50 μg/mL. The lower limit of quantification was 0.1 μg/mL. The intra‐ and inter‐day precisions and the accuracy all satisfied the acceptance criteria. The developed method was validated and successfully applied to the pharmacokinetics study of DS aqueous injection and LM injection. The results showed that the two preparations were bioequivalent in rats. Copyright © 2009 John Wiley & Sons, Ltd.