Quantitative ultra‐high‐performance liquid chromatography–tandem mass spectrometry for determination of dexmedetomidine in pediatric plasma samples: Correlation with genetic polymorphisms

Dexmedetomidine is an important sedative agent administered as premedication to achieve procedural sedation in children. To describe the correlation between the genetic state and the concentration of dexmedetomidine, it is necessary to develop a specific, time‐saving and economical method for detection of dexmedetomidine in plasma samples. In this work, an ultra‐high‐performance liquid chromatography (UHPLC)–tandem mass spectrometry method has been established and validated for detection of dexmedetomidine in plasma from pediatric population. After a simple liquid–liquid extraction with an organic solution, the analytes were separated on an ACQUITY BEH C₁₈ column (2.1 mm × 50 mm, 1.7 μm particle size) by gradient elution with the mobile phase of acetonitrile and 1‰ aqueous formic acid (flow rate 0.3 mL min^?1). Mass spectrometry measurements were performed under the positive selected reaction monitoring and the mass transitions monitored were m/z 201.3 → 95.1, 204.2 → 98.0 for dexmedetomidine and deuterated medetomidine (internal standard), respectively. Validation of the method based on China Food and Drug Administration guidelines showed acceptable selectivity. The UHPLC method employed a stable isotope‐labeled internal standard, showed good specificity and was successfully used to detect dexmedetomidine in plasma samples from 260 pediatric patients. A subsequent application of this method to a pharmacogenetic study was also described. Importantly, this is the first study to report the correlation between CYP2A6 rs835309 activity and concentration of dexmedetomidine.     

An improved ultra‐high‐performance liquid chromatography–tandem mass spectrometric method for the quantitation of dexmedetomidine in small volume of pediatric plasma

Dexmedetomidine (Dex), a highly selective α₂‐adrenergic agonist, is used primarily for the sedation and anxiolysis of adults and children in the intensive care setting. A sensitive and selective assay for Dex in pediatric plasma was developed by employing ultra‐high‐performance liquid chromatography–tandem mass spectrometry with d4‐Dex as an internal standard. Dex was extracted from 0.1 mL of plasma by micro‐elution solid‐phase extraction. Separation was achieved with a Waters XBridge C₁₈ column with a flow rate of 0.3 mL/min using a mobile phase comprising 5 mm ammonium acetate buffer with 0.03% formic acid in water and methanol–acetonitrile (50:50, v/v). The intra‐day precision (coefficient of variation) and accuracy for quality control samples ranged from 1.32 to 8.91% and from 92.8 to 108%, respectively. The inter‐day precision and accuracy ranged from 2.13 to 8.45% and from 97.0 to 104%, respectively. The analytical method showed excellent sensitivity using a small sample volume (0.1 mL) with a lower limit of quantitation of 5 pg/mL. This method is robust and has been successfully employed in a pharmacokinetic study of Dex in neonates and infants postoperative from cardiac surgery.     

Ultra‐high pressure liquid chromatography–tandem mass spectrometry method for the determination of omarigliptin in rat plasma and its application to a pharmacokinetic study in rats

Omarigliptin is a novel long‐acting dipeptidyl peptidase‐4 inhibitor used for the treatment of type 2 diabetes. In this work, a sensitive and selective ultra‐high pressure liquid chromatography tandem mass spectrometry method was developed and validated for the determination of omarigliptin in rat plasma. Sample preparation was performed by protein precipitation with acetonitrile. Chromatographic separation of analytes was achieved on an RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm), using gradient mobile phase (0.1% formic acid–acetonitrile) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode, with target fragment ions m/z 399.1 → 152.9 for omarigliptin and m/z 237.1 → 194 for the internal standard. The total run time was 4 min. Retention time of omarigliptin and internal standard was 1.25 and 2.12 min, respectively. Relative standard deviation (%) of the intra‐ and inter‐day precision was below 10.0%, and accuracy was between 97.9% and 105.3%. Calibration curve was established over the range 2–5000 ng/mL with good linearity. The lower limit of quantification and limit of detection of omarigliptin were 2 and 0.25 ng/mL, respectively. Mean recoveries were in the range 87.3–95.1% for omarigliptin. No matrix effect was observed in this method. This novel method has been successfully applied to a pharmacokinetic study of omarigliptin in rats. The absolute bioavailability of omarigliptin was identified as high as 87.31%.     

Analysis of carbenoxolone by ultra‐high‐performance liquid chromatography tandem mass spectrometry in mouse brain and blood after systemic administration

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high‐performance liquid chromatography tandem mass spectrometer (UHPLC–MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half‐lives in mice of ~43 and 41 min, respectively. The UHPLC–MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.     

Simultaneous determination of tilianin and its metabolites in mice using ultra‐high‐performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Tilianin is an active flavonoid glycoside found in many medical plants. Data are lacking regarding its pharmacokinetics and disposition in vivo. The objective of this study was to develop a sensitive, reliable and validated ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method to simultaneously quantify tilianin and its main metabolites and to determine its pharmacokinetics in wild‐type and breast cancer resistance protein knockout (Bcrp1−/−) FVB mice. Chromatographic separation was accomplished on a C₁₈ column by utilizing acetonitrile and 0.5 mm ammonium acetate as the mobile phase. Mass spectrometric detection was performed using electrospray ionization in both positive and negative modes. The results showed that the precision, accuracy and recovery, as well as the stability of tilianin and its metabolites in mouse plasma, were all within acceptable limits. Acacetin‐7‐glucuronide and acacetin‐7‐sulfate were the major metabolites of tilianin in mouse plasma. Moreover, systemic exposure of acacetin‐7‐sulfate was significantly higher in Bcrp1 (−/−) FVB mice compared with wild‐type FVB mice. In conclusion, the fully validated UHPLC–MS/MS method was sensitive, reliable, and was successfully applied to assess the pharmacokinetics of tilianin in wild‐type and Bcrp1 (−/−) FVB mice. Breast cancer resistance protein had a significant impact on the elimination of the sulfated metabolite of tilianin in vivo.     

Liquid chromatography–tandem mass spectrometry method of loxoprofen in human plasma

A rapid, sensitive and selective liquid chromatography–electrospray ionization mass spectrometric method for the determination of loxoprofen in human plasma was developed. Loxoprofen and ketoprofen (internal standard) were extracted from 20 µL of human plasma sample using ethyl acetate at acidic pH and analyzed on an Atlantis dC₁₈ column with the mobile phase of methanol:water (75:25, v/v). The analytes were quantified in the selected reaction monitoring mode. The standard curve was linear over the concentration range of 0.1–20 µg/mL with a lower limit of quantification of 0.1 µg/mL. The coefficient of variation and relative error for intra‐ and inter‐assay at four quality control levels were 2.8–5.2 and 4.8–7.0%, respectively. The recoveries of loxoprofen and ketoprofen were 69.7 and 67.6%, respectively. The matrix effects for loxoprofen and ketoprofen were practically absent. This method was successfully applied to the pharmacokinetic study of loxoprofen in humans. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous quantification of oxybutynin and its active metabolite N‐desethyl oxybutynin in rat plasma by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study of oxybutynin transdermal patch

A rapid, selective and sensitive ultra‐high‐performance liquid chromatography–tandem mass spectrometry method was developed to simultaneously determine oxybutynin and its active metabolite N‐desethyl oxybutynin in rat plasma. A 0.1 mL sample of plasma was extracted with n‐hexane. Chromatographic separation was performed on a UPLC BEH C₁₈ column (2.1 × 100 mm i.d.,1.7 μm) with mobile phase of methanol–water (containing 2 mmol/L ammonium acetate and 0.1% formic acid; 90:10, v/v). The detection was performed in positive selected reaction monitoring mode. Each plasma sample was chromatographed within 3 min. The linear calibration curves were obtained in the concentration range of 0.0944–189 ng/mL (r ≥ 0.99) for oxybutynin and 0.226–18.0 ng/mL (r ≥ 0.99) for N‐desethyl oxybutynin. The intra‐ and inter‐day precision (relative standard deviation) values were not more than 14% and the accuracy (relative error) was within ±7.6%. The method described was superior to previous methods for the quantitation of oxybutynin with three product ions and was successfully applied to a pharmacokinetic study of oxybutynin and its active metabolite N‐desethyl oxybutynin in rat plasma after transdermal administration.     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of l‐tetrahydropalmatine and its active metabolites in rat plasma by a sensitive ultra‐high‐performance liquid chromatography with tandem mass spectrometry method and its application in a pharmacokinetic study

A sensitive and reliable ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of l ‐tetrahydropalmatine (l ‐THP) and its active metabolites l ‐isocorypalmine (l ‐ICP) and L ‐corydalmine (l ‐CD) in rat plasma. The analytes were extracted by liquid–liquid extraction and separated on a Bonshell ASB C₁₈ column (2.1 × 100 mm; 2.7 μm; Agela) using acetonitrile–formic acid aqueous as mobile phase at a flow rate of 0.2 mL/min in gradient mode. The method was validated over the concentration range of 4.00–2500 ng/mL for l ‐THP, 0.400–250 ng/mL for l ‐ICP and 1.00–625 ng/mL for l ‐CD. Intra‐ and inter‐day accuracy and precision were within the acceptable limits of <15% at all concentrations. Correlation coefficients (r ) for the calibration curves were >0.99 for all analytes. The quantitative method was successfully applied for simultaneous determination of l ‐THP and its active metabolites in a pharmacokinetic study after oral administration with l ‐THP at a dose of 15 mg/kg to rats.     

Ultra‐performance liquid chromatography–tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women

A highly sensitive and selective ultra‐performance liquid chromatography–tandem mass spectrometry method is described for the simultaneous determination of nomegestrol acetate (NOMAC), a highly selective progestogen, and estradiol (E2), a natural estrogen in human plasma. NOMAC was obtained from plasma by solid‐phase extraction, while E2 was first separated by liquid–liquid extraction with methyl tert‐butyl ether followed by derivatization with dansyl chloride. Deuterated internal standards, NOMAC‐d5 and E2‐d4 were used for better control of extraction conditions and ionization efficiency. The assay recovery of the analytes was within 90–99%. The analytes were separated on UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using a mobile phase comprising of acetonitrile and 3.0 mm ammonium trifluoroacetate in water (80:20, v/v) with a resolution factor (R_s) of 3.21. The calibration curves were linear from 0.01 to 10.0 ng/mL for NOMAC and from 1.00 to 1000 pg/mL for E2, respectively. The intra‐ and inter‐batch precision was ≤5.8% and the accuracy of quality control samples ranged from 96.7 to 103.4% for both analytes. The practical applicability of the method is demonstrated by analyzing samples from 18 healthy postmenopausal women after oral administration of 2.5 mg nomegestrol acetate and 1.5 mg estradiol film‐coated tablets under fasting.     

Quantification and pharmacokinetic study of entrectinib in rat plasma using ultra‐performance liquid chromatography tandem mass spectrometry

To characterize the preclinical plasma pharmacokinetics of entrectinib, a reproducible and precise assay is necessary. In this study, we developed and validated a simple ultra‐performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the measurement of entrectinib using carbamazepine as the internal standard in rat plasma. Sample preparation was a simple protein precipitation with acetonitrile, then entrectinib was eluted on an Acquity UPLC BEH C₁₈ column (2.1 × 50 mm, 1.7 μm) using a gradient elution with a mobile phase composed of acetonitrile (A) and 0.1% formic acid in water (B). Detection was achieved using multiple‐reaction monitoring in positive ion electrospray ionization mode. The method showed good linearity over the concentration range of 1–250 ng/mL (r² > 0.9951). The intra‐ and inter‐day precision was determined with the values of 6.3–12.9 and 2.6–6.9%, respectively, and accuracy values of 0.5–11.6%. Matrix effect, extraction recovery, and stability data all met the acceptance criteria of US Food and Drug Administration guidelines for bioanalytical method validation. The method was successfully applied to a pharmacokinetic study. In this study, we developed the complete validated method for the quantification of entrectinib in rat plasma.     

Determination of mycophenolic acid in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its pharmacokinetic application in kidney transplant patients

To implement and validate an analytical method by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC MS/MS) to quantify mycophenolic acid (MPA) in kidney transplant patients. Quantification of MPA was performed in an ACQUITY UPLC H Class system coupled to a Xevo TQD detector and it was extracted from plasma samples by protein precipitation. The chromatographic separation was achieved through an ACQUITY HSS C₁₈ SB column with 0.1% formic acid and acetonitrile (60:40 vol/vol) as mobile phase. The pharmacokinetic parameters were calculated by non‐compartmental analysis of MPA plasma concentrations from 10 kidney transplant patients. The linear range for MPA quantification was 0.2–30 mg/L with a limit of detection of 0.07 mg/L; the mean extraction recovery was 99.99%. The mean intra‐ and inter‐day variability were 2.98% and 3.4% with a percentage of deviation of 8.4% and 6.6%, respectively. Mean maximal concentration of 10 mg/L at 1.5 h, area under the concentration–time curve of 36.8 mg·h/L, elimination half‐life of 3.9 h, clearance of 0.32 L/h/kg and volume of distribution of 1.65 L/kg were obtained from MPA pharmacokinetics profiles. A simple, fast and reliable UPLC–MS/MS method to quantify MPA in plasma was validated and has been applied for pharmacokinetic analysis in kidney transplant patients.     

A high‐performance liquid chromatography–tandem mass spectrometry method coupled with protein precipitation for determination of granisetron in human plasma and its application to a comparative pharmacokinetic study

A rapid, simple and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) has been developed for the determination of granisetron in human plasma. Plasma samples were pre‐purified by protein precipitation procedure. The chromatographic separation was achieved with Synergi Polar‐RP (75 × 2 mm, 4 µm) column using a mixture of 5 mm pH4.0 ammonium formate and methanol (300:316, v/v) under isocratic conditions at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The analysis time was about 2.5 min. The method was fully validated over the concentration range 0.1–10 ng/mL. The lower limit of quantification was 0.1 ng/mL. Inter‐ and intra‐batch precision was <6.1% and the accuracy was within 95.6–100.0%. The mean extraction recovery was 96.3%. Selectivity, matrix effect and stability were also validated. The method was applied to the comparative pharmacokinetic study of granisetron in Chinese healthy subjects. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

Determination of phenamacril residues in flour and rice based on Z‐Sep+ using ultra‐high‐performance liquid chromatography–tandem mass spectrometry

Phenamacril is a new broad‐spectrum fungicide that is commonly used for the control of fungal diseases in wheat and rice. In this study, ultra‐high‐performance liquid chromatography–tandem mass spectrometry was used to establish a method for analyzing the residual phenamacril in flour and rice based on the improved QuEChERS (quick, easy, cheap, effective, rugged and safe) method using Z‐Sep+ as the adsorbent in the pre‐treatment process. The average recovery of phenamacril in flour and rice was 82.2–96.0%, the relative standard deviation was 2.1–5.6% and the limit of quantification was 0.5 μg/kg. The accuracy and sensitivity of this method meet the requirements for residue analysis. The method was applied to commercially available flour and rice samples, and the detected concentrations of phenamacril were 0.005–0.033 mg/kg. This method provides technical support for the safety evaluation of phenamacril.     

Simultaneous ultra‐performance liquid chromatography–tandem mass spectrometry determination of six components in rat plasma after oral administration of Smilacis glabrae Roxb. extract

In this study, an accurate and reliable method of ultra‐performance liquid chromatography coupled with a triple‐quadrupole tandem mass spectrometry was firstly developed and fully validated for the simultaneous determination of epicatechin, neoastilbin, astilbin, isoastilbin, engeletin and resveratrol in rat plasma after administration of Smilacis glabrae Roxb. extract. Naringenin was used as an internal standard (IS). The analyte and IS were separated on a C₁₈ column by gradient elution with a mobile phase of acetonitrile–0.3% acetic acid at a flow rate of 0.25 mL/min for a total run time of 8 min. The method was validated in terms of selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability. The developed method was successfully applied to determine the main pharmacokinetic parameters of six components in rat plasma.     

Simultaneous determination of ambroxol and salbutamol in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A rapid, selective and sensitive liquid chromatography–tandem mass spectrometry assay method was developed for simultaneous determination of ambroxol and salbutamol in human plasma using citalopram hydrobromide as internal standard (IS). The sample was alkalinized with ammonia water (33:67, v/v) and extracted by single liquid–liquid extraction with ethyl acetate. Separation was achieved on Waters Acquity UPLC BEH C₁₈ column using a gradient program at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 378.9 → 263.6 (ambroxol), m/z 240.2 → 147.7 (salbutamol) and m/z 325.0 → 261.7 (IS). The total analytical run time was relatively short (3 min). Calibration curves were linear in the concentration range of 0.5–100.0 ng/mL for ambroxol and 0.2–20.0 ng/mL for salbutamol, with intra‐ and inter‐run precision (relative standard deviation) <15% and accuracy (relative error) ranging from 97.7 to 112.1% for ambroxol and from 94.5 to 104.1% for salbutamol. The method was successfully applied in a clinical pharmacokinetic study of the compound ambroxol and salbutamol tablets.     

Carboxylesterase and UDP‐glucuronosyltransferases mediated metabolism of irinotecan: In vitro and in vivo insights from quantitative ultra‐performance liquid chromatography–mass spectrometry analysis

Carboxylesterase and UDP‐glucuronosyltransferase‐mediated metabolism of irinotecan (CPT‐11) has long been proposed to be responsible for its anti‐tumor activity and toxicity, like delayed‐onset diarrhea. However, recent studies failed to gain more comprehensive in vivo and in vitro pharmacokinetic profiles of irinotecan. Herein, we use rat plasma, human liver microsomes and immortalized HepG2 cell as experimental subjects to describe a sensitive and versatile UHPLC–MS/MS method for simultaneously quantifying CPT‐11 and its metabolites, including SN‐38 and SN‐38G. The method was applied to investigate the pharmacokinetic and metabolic behavior of CPT‐11 in the biological samples. Calibration curves for all bio‐matrices showed acceptable linearity (r² > 0.99). The intra‐ and inter‐day precisions (RSD, %) were within 15% and the excellent accuracy (RE) was between 2.96 and 14.12%. In addition, the specificity, matrix effect and extraction recovery all met the requirements of biological sample analysis. We successfully applied this method to investigate the pharmacokinetics of irinotecan in various biological samples, mediated by carboxylesterase and UDP‐glucuronosyltransferase. This method could be employed in monitoring the metabolic status and clinical efficacy of irinotecan in the future.     

Simultaneous quantification of metformin and glipizide in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A selective, sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method was developed and validated to determine metformin and glipizide simultaneously in human plasma using phenacetin as internal standard (IS). After one‐step protein precipitation of 200 μL plasma with methanol, metformin, glipizide and IS were separated on a Kromasil Phenyl column (4.6 × 150 mm, 5 µm) at 40°C with an isocratic mobile phase consisting of methanol–10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.35 mL/min. Electrospray ionization source was applied and operated in the positive mode. Multiple reaction monitoring using the precursor → product ion combinations of m/z 130 → m/z 71, m/z 446 → m/z 321 and m/z 180 → m/z 110 were used to quantify metformin, glipizide and IS, respectively. The linear calibration curves were obtained over the concentration ranges 4.10–656 ng/mL for metformin and 2.55–408 ng/mL for glipizide. The relative standard deviation of intra‐day and inter‐day precision was below 10% and the relative error of accuracy was between ?7.0 and 4.6%. The presented HPLC‐MS/MS method was proved to be suitable for the pharmacokinetic study of metformin hydrochloride and glipizide tablets in healthy volunteers after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid assay for angiotensin‐converting enzyme activity using ultra‐performance liquid chromatography–mass spectrometry

Angiotensin‐converting enzyme (ACE) plays an important role in the renin–angiotensin system and ACE activity is usually assayed in vitro by monitoring the transformation from a substrate to the product catalyzed by ACE. A rapid and sensitive analysis method or ACE activity by quantifying simultaneously the substrate hippuryl–histidyl–leucine and its product hippuric acid using an ultra‐performance liquid chromatography coupled with electrospray ionization‐mass spectrometry (UPLC‐MS) was first developed and applied to assay the inhibitory activities against ACE of several natural phenolic compounds. The established UPLC‐MS method showed obvious advantages over the conventional HPLC analysis in shortened running time (3.5 min), lower limit of detection (5 pg) and limit of quantification (18 pg), and high selectivity aided by MS detection in selected ion monitoring (SIM) mode. Among the six natural products screened, five compounds, caffeic acid, caffeoyl acetate, ferulic acid, chlorogenic acid and resveratrol indicated potent in vitro ACE inhibitory activity with IC₅₀ values of 2.527 ± 0.032, 3.129 ± 0.016, 10.898 ± 0.430, 15.076 ± 1.211 and 6.359 ± 0.086 mm , respectively. A structure–activity relationship estimation suggested that the number and the situation of the hydroxyls on the benzene rings and the acrylic acid groups may play the most predominant role in their ACE inhibitory activity. Copyright © 2009 John Wiley & Sons, Ltd.