Geographical discrimination of Glechomae Herba based on fifteen phenolic constituents determined by LC–MS/MS method combined with chemometric methods

Glechomae Herba (GH) is rich in bioactive phenolic constituents, which is widely used for treatment of cholelithiasis, urolithiasis and dropsy. The simultaneous determination of phenolic constituents in GH from different geographical origins is significant for authentication and quality control purposes. In this study, we developed a strategy integrating targeted analysis and chemometric methods for quality evaluation and discrimination of GH from different geographical origins. Firstly, an accurate and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of fifteen phenolic constituents in GH from different geographical origins. The established method was well validated in terms of desirable specificity, linearity, precision and accuracy. Secondly, the quantitative data were subjected to the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Thirdly, a heatmap visualization was employed for clarifying the distribution of fifteen phenolic compounds in GH from different geographical origins. These results indicated that GH samples from Shandong province have obviously different with those from other provinces in the content of bioactive phenolic constituents. Collectively, the proposed platform might be well-acceptable tool for quality evaluation and discrimination of GH from different geographical origins, providing promising perspectives in tracking the formulation processes of TCMs products.

Abstract

Glechomae Herba (GH) is rich in bioactive phenolic constituents, which is widely used for treatment of cholelithiasis, urolithiasis and dropsy. The simultaneous determination of phenolic constituents in GH from different geographical origins is significant for authentication and quality control purposes. In this study, we developed a strategy integrating targeted analysis and chemometric methods for quality evaluation and discrimination of GH from different geographical origins. Firstly, an accurate and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of fifteen phenolic constituents in GH from different geographical origins. The established method was well validated in terms of desirable specificity, linearity, precision and accuracy. Secondly, the quantitative data were subjected to the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Thirdly, a heatmap visualization was employed for clarifying the distribution of fifteen phenolic compounds in GH from different geographical origins. These results indicated that GH samples from Shandong province have obviously different with those from other provinces in the content of bioactive phenolic constituents. Collectively, the proposed platform might be well-acceptable tool for quality evaluation and discrimination of GH from different geographical origins, providing promising perspectives in tracking the formulation processes of TCMs products.

Detection of trace amounts of citrinin in dried orange peel by using an optimized extraction method coupled with ultra-performance liquid chromatography–tandem mass spectrometry

A fast and sensitive method involving ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was introduced to detect citrinin in dried orange peel. A series of extraction, purification, and chromatographic conditions was also systematically examined. With the proposed method, the obtained calibration graph was linear, with an R of 0.9996 within a concentration range of 0.5–10 ng/mL. The estimated limits of detection and quantification were 0.05 and 0.17 ng/mL, respectively. Under the selected conditions, the relative recoveries in different citrus products spiked with 1–10 ng/mL citrinin were 89.4%–98.7% with RSDs of below 2.5%. Compared with previously reported analytical methods, the newly developed UPLC-MS/MS method showed excellent sensitivity and good precision in detecting citrinin. Results indicated that it is a reliable and effective technique for the detection of trace citrinin in dried orange peel.

Abstract

A fast and sensitive method involving ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was introduced to detect citrinin in dried orange peel. A series of extraction, purification, and chromatographic conditions was also systematically examined. With the proposed method, the obtained calibration graph was linear, with an R of 0.9996 within a concentration range of 0.5–10 ng/mL. The estimated limits of detection and quantification were 0.05 and 0.17 ng/mL, respectively. Under the selected conditions, the relative recoveries in different citrus products spiked with 1–10 ng/mL citrinin were 89.4%–98.7% with RSDs of below 2.5%. Compared with previously reported analytical methods, the newly developed UPLC-MS/MS method showed excellent sensitivity and good precision in detecting citrinin. Results indicated that it is a reliable and effective technique for the detection of trace citrinin in dried orange peel.

In vivo metabolite profiles of isoimperatorin and phellopterin in rats analyzed by using HPLC coupled with diode array detector and electrospray ionization ion trap time-of-flight mass spectrometry technique

Isoimperatorin (IP) and phellopterin (PP) are two furocoumarins existed in Angelicae Dahuricae Radix, there is an isopentenyloxyl substituted at C-5 in IP, while an isopentenyloxyl and a methoxyl substituted at C-8 and C-5 in PP, respectively. To elucidate the in vivo metabolic characteristics of PP and IP, HPLC coupled with diode array detector and electrospray ionization ion trap time-of-flight mass spectrometry technique was used. Totally, 110 metabolites, including 53 new ones, were identified from the urine and plasma samples of rats after oral administration of IP and PP, respectively. The metabolites were formed through 8 reactions on IP and PP, including oxidation, hydroxylation-hydrogenation, carboxylation on the isopentenyloxyl; O-dealkylation, hydroxylation on the furocoumarin nucleus; ring-opening reaction on the furan ring; and reduction or ring-opening reaction on the lactone ring. Among which, hydroxylation on the furocoumarin nucleus was first found for in vivo metabolites of PP and IP, ring-opening reaction on the furan ring or lactone ring were first found for in vivo metabolites of isopentenyloxyl furocoumarins. The research gave us a new insight on the in vivo metabolic profiles of IP and PP, which could help us better understand their important roles for being two active constituents of Angelicae Dahuricae Radix.

Abstract

Isoimperatorin (IP) and phellopterin (PP) are two furocoumarins existed in Angelicae Dahuricae Radix, there is an isopentenyloxyl substituted at C-5 in IP, while an isopentenyloxyl and a methoxyl substituted at C-8 and C-5 in PP, respectively. To elucidate the in vivo metabolic characteristics of PP and IP, HPLC coupled with diode array detector and electrospray ionization ion trap time-of-flight mass spectrometry technique was used. Totally, 110 metabolites, including 53 new ones, were identified from the urine and plasma samples of rats after oral administration of IP and PP, respectively. The metabolites were formed through 8 reactions on IP and PP, including oxidation, hydroxylation-hydrogenation, carboxylation on the isopentenyloxyl; O-dealkylation, hydroxylation on the furocoumarin nucleus; ring-opening reaction on the furan ring; and reduction or ring-opening reaction on the lactone ring. Among which, hydroxylation on the furocoumarin nucleus was first found for in vivo metabolites of PP and IP, ring-opening reaction on the furan ring or lactone ring were first found for in vivo metabolites of isopentenyloxyl furocoumarins. The research gave us a new insight on the in vivo metabolic profiles of IP and PP, which could help us better understand their important roles for being two active constituents of Angelicae Dahuricae Radix.

Simultaneous determination of three flavonoids and one coumarin by LC-MS/MS: application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Daphne genkwa extract

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin, and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple-quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161133 for umbelliferone, m/z 269117 for apigenin, m/z 283268 for genkwanin, and m/z 299284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB-C18 column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421–1421 ng/mL for umbelliferone, 0.845–845 ng/mL for apigenin, 1.025–1025 ng/mL for genkwanin, and 0.845–845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats.

Abstract

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin, and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple-quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161[RIGHTWARDS ARROW]133 for umbelliferone, m/z 269[RIGHTWARDS ARROW]117 for apigenin, m/z 283[RIGHTWARDS ARROW]268 for genkwanin, and m/z 299[RIGHTWARDS ARROW]284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB-C18 column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421–1421 ng/mL for umbelliferone, 0.845–845 ng/mL for apigenin, 1.025–1025 ng/mL for genkwanin, and 0.845–845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats.

Preparation of dummy template-imprinted polymers for the rapid extraction of nonsteroidal anti-inflammatory drugs residues in aquatic environmental samples

A molecularly imprinted polymer was synthesized and applied as a sorbent in the solid-phase extraction device. The imprinted polymer was characterized by fourier-transform infrared spectroscopy and scanning electron microscope. The results revealed that imprinted polymer possess sensitive selectivity and reliable adsorption properties for five NSAIDs. The imprinted polymer was successfully applied to the pre-concentration for five NSAIDs in different water samples prior to UPLC-MS/MS. In the early studies, several factors were investigated, including pH adjustment, the kind of elution solvent and the volume of elution solvent. Finally, we found that the pH 5 and an aliquot of 2 mL methanol were suitable for the water samples. The limits of detection and limits of quantitation of five nonsteroidal anti-inflammatory drugs varied from 0.007 to 0.480 μg L−1 and 0.03 to 1.58 μg L−1, respectively. The spiking recoveries of the target analytes were 50.33-127.64% at the levels of 0.2 μg L−1, 2 μg L−1 and 5 μg L−1. The precision and accuracy of this method showed a great increase compared with traditional solid-phase extraction. The developed method was successfully applied to extraction and analysis of NSAIDs in different water samples with satisfactory results which could help us better understand their environmental fate and risk to ecological health.

Abstract

A molecularly imprinted polymer was synthesized and applied as a sorbent in the solid-phase extraction device. The imprinted polymer was characterized by fourier-transform infrared spectroscopy and scanning electron microscope. The results revealed that imprinted polymer possess sensitive selectivity and reliable adsorption properties for five NSAIDs. The imprinted polymer was successfully applied to the pre-concentration for five NSAIDs in different water samples prior to UPLC-MS/MS. In the early studies, several factors were investigated, including pH adjustment, the kind of elution solvent and the volume of elution solvent. Finally, we found that the pH 5 and an aliquot of 2 mL methanol were suitable for the water samples. The limits of detection and limits of quantitation of five nonsteroidal anti-inflammatory drugs varied from 0.007 to 0.480 μg L−1 and 0.03 to 1.58 μg L−1, respectively. The spiking recoveries of the target analytes were 50.33-127.64% at the levels of 0.2 μg L−1, 2 μg L−1 and 5 μg L−1. The precision and accuracy of this method showed a great increase compared with traditional solid-phase extraction. The developed method was successfully applied to extraction and analysis of NSAIDs in different water samples with satisfactory results which could help us better understand their environmental fate and risk to ecological health.

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 304.1161.1 for evodiamine, m/z 320.1134.1 for M1, m/z 320.1150.1 for M2, m/z 496.2134.1 for M3, m/z 496.2171.1 for M4 and m/z 349.2305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients greater than 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 less than 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study would be helpful in understanding the in vivo disposition of evodiamine.

Abstract

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[RIGHTWARDS ARROW]161.1 for evodiamine, m/z 320.1[RIGHTWARDS ARROW]134.1 for M1, m/z 320.1[RIGHTWARDS ARROW]150.1 for M2, m/z 496.2[RIGHTWARDS ARROW]134.1 for M3, m/z 496.2[RIGHTWARDS ARROW]171.1 for M4 and m/z 349.2[RIGHTWARDS ARROW]305.1 for camptothecin (internal standard). The linearity was evident over the tested concentration ranges with correlation coefficients greater than 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 less than 9.31%. The validated assay was successfully applied for the pharmacokinetic study of evodiamine, M1, M2, M3 and M4 in rat. The current study would be helpful in understanding the in vivo disposition of evodiamine.

Determination of homogentisic acid in urine for diagnosis of alcaptonuria: Capillary electrophoretic method optimization using experimental design

Homogentisic acid (HGA) is a diagnostic metabolite that accumulates in the urine and tissues of patients with alkaptonuria which is a rare autosomal recessive disease. HGA is a specific metabolite in urine and serum, which is used for diagnosis of alkaptonuria. This study presents an inexpensive and easy capillary electrophoretic (CE) method for the quantitative determination of HGA in urine samples. CE method were optimized using full factorial experimental design. The optimal separation electrolyte and separation voltage were revealed as 45 mmol/L phosphate buffer at pH 7.0 and 22 kV, respectively. Under these conditions the presence of HGA is detected in 6 min. Repeatability of migration times and corrected peak areas of HGA (as R.S.D.%) were found as 0.37 and 1.99, respectively. The detection limit (LOD) was 0.56 μg/mL as 3 times of the average noise and the quantification limit (LOQ) was 1.85 μg/mL as 10 times of the average noise for HGA. Urine samples were directly injected to the capillary without any pretreatment step.

Abstract

Homogentisic acid (HGA) is a diagnostic metabolite that accumulates in the urine and tissues of patients with alkaptonuria which is a rare autosomal recessive disease. HGA is a specific metabolite in urine and serum, which is used for diagnosis of alkaptonuria. This study presents an inexpensive and easy capillary electrophoretic (CE) method for the quantitative determination of HGA in urine samples. CE method were optimized using full factorial experimental design. The optimal separation electrolyte and separation voltage were revealed as 45 mmol/L phosphate buffer at pH 7.0 and 22 kV, respectively. Under these conditions the presence of HGA is detected in 6 min. Repeatability of migration times and corrected peak areas of HGA (as R.S.D.%) were found as 0.37 and 1.99, respectively. The detection limit (LOD) was 0.56 μg/mL as 3 times of the average noise and the quantification limit (LOQ) was 1.85 μg/mL as 10 times of the average noise for HGA. Urine samples were directly injected to the capillary without any pretreatment step.

Characterization of Neuropeptide K Processing in Rat Spinal Cord S9 Fractions using High-Resolution Quadrupole-Orbitrap Mass Spectrometry

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA were very short resulting in half-lives of 1.9 and 2.2 minutes respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK1-26, NKA, NKA2-10, NKA3-10, NKA5-10 and NKA6-10, which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA providing strong evidence that proprotein convertases is involved into C-terminal processing of NPK in the spinal cord leading to the formation of NKA.

Abstract

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA were very short resulting in half-lives of 1.9 and 2.2 minutes respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK1-26, NKA, NKA2-10, NKA3-10, NKA5-10 and NKA6-10, which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA providing strong evidence that proprotein convertases is involved into C-terminal processing of NPK in the spinal cord leading to the formation of NKA.

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 AUCBrain/AUCBlood 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.

Abstract

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 AUCBrain/AUCBlood 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.

Metabolite identification of the antimalarial naphthoquine using liquid chromatography–tandem high-resolution mass spectrometry in combination with multiple data-mining tools

Naphthoquine (NQ) is one of important partner drugs of artemisinin-based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half-life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMSn) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid-phase extraction. For data processing, multiple data-mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N-oxidation. This study demonstrates that LC-HRMSn in combination with multiple data-mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.

Abstract

Naphthoquine (NQ) is one of important partner drugs of artemisinin-based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half-life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMSn) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid-phase extraction. For data processing, multiple data-mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N-oxidation. This study demonstrates that LC-HRMSn in combination with multiple data-mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.

Supramolecular separation mechanism of pentafluorophenyl column using ibuprofen and omeprazole as markers: LC-MS and simulation study

The pentafluorophenyl (PFP) column is emerging as a new advancement in separation science to analyze a wide range of analytes and, thus, its separation mechanism at supramolecular level is significant. We developed a mechanism for the separation of ibuprofen and omeprazole using different combinations (ranging from 50:50 to 60:40) of water–acetonitrile containing 0.1% formic acid as the mobile phase. The column used was Waters Acquity UPLC HSS PFP (75 × 2.1 mm, 1.8 μm). The reverse order of elution was observed in different combinations of the mobile phases. The docking study indicated hydrogen bonding between ibuprofen and PFP stationary phase (binding energy was −11.30 kJ/mol). Separation at PFP stationary phase is controlled by hydrogen bonding along with π–π interactions. This stationary phase may be used to analyze both aromatic and aliphatic analytes. The developed mechanism will be useful to separate various analytes by considering the possible interactions, leading to saving of energy, time and money. In addition, this work will be highly useful in preparative chromatography where separation is the major problem at a large scale. Moreover, the developed LC-MS-QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample owing to the low value of detection limits.

Abstract

The pentafluorophenyl (PFP) column is emerging as a new advancement in separation science to analyze a wide range of analytes and, thus, its separation mechanism at supramolecular level is significant. We developed a mechanism for the separation of ibuprofen and omeprazole using different combinations (ranging from 50:50 to 60:40) of water–acetonitrile containing 0.1% formic acid as the mobile phase. The column used was Waters Acquity UPLC HSS PFP (75 × 2.1 mm, 1.8 μm). The reverse order of elution was observed in different combinations of the mobile phases. The docking study indicated hydrogen bonding between ibuprofen and PFP stationary phase (binding energy was −11.30 kJ/mol). Separation at PFP stationary phase is controlled by hydrogen bonding along with π–π interactions. This stationary phase may be used to analyze both aromatic and aliphatic analytes. The developed mechanism will be useful to separate various analytes by considering the possible interactions, leading to saving of energy, time and money. In addition, this work will be highly useful in preparative chromatography where separation is the major problem at a large scale. Moreover, the developed LC-MS-QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample owing to the low value of detection limits.

Simultaneous Measurement of NAD Metabolome in Aged Mice Tissue Using Liquid Chromatography Tandem-Mass Spectrometry (LC/MS/MS)

Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels declined with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on a LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH by performing a single sample preparation. Further, we validate our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase of in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.

Abstract

Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels declined with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on a LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH by performing a single sample preparation. Further, we validate our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase of in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.

Simultaneous determination of gelsemine and koumine in rat plasma by UPLC-MS/MS and application to pharmacokinetic study after oral administration of Gelsemium elegans Benth extract

A simple, rapid and sensitive method using UPLC-MS/MS was established and validated for simultaneous determination of gelsemine and koumine in rat plasma after oral administration of Gelsemium elegans Benth extract. Plasma was performed with methanol precipitation and berberine was chosen as the internal standard. Plasma samples were separated on an Acquity UPLC® BEH C18 column (3.0 × 50 mm, 1.7 μm) with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring mode in positive ion mode was utilized for detection. The calibration curves were linear over the range of 0.2–100 ng/mL for gelsemine and 0.1–50 ng/mL for koumine, with the lower limits of quantification 0.2 and 0.1 ng/mL, respectively. The intra- and inter-precision and accuracy were well within the acceptable ranges. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10 mg/kg Gelsemium elegans Benth extract.

Abstract

A simple, rapid and sensitive method using UPLC-MS/MS was established and validated for simultaneous determination of gelsemine and koumine in rat plasma after oral administration of Gelsemium elegans Benth extract. Plasma was performed with methanol precipitation and berberine was chosen as the internal standard. Plasma samples were separated on an Acquity UPLC® BEH C18 column (3.0 × 50 mm, 1.7 μm) with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring mode in positive ion mode was utilized for detection. The calibration curves were linear over the range of 0.2–100 ng/mL for gelsemine and 0.1–50 ng/mL for koumine, with the lower limits of quantification 0.2 and 0.1 ng/mL, respectively. The intra- and inter-precision and accuracy were well within the acceptable ranges. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10 mg/kg Gelsemium elegans Benth extract.

Monitoring pesticide residues in dates marketed in Al-Qassim, Saudi Arabia using a QuEChERS methodology and liquid chromatography-tandem mass spectrometry

A sensitive, simple and rapid QuEChERS extraction method and liquid chromatography equipped with triple quadrupole mass spectrometry (LC-MS/MS) were used to evaluate 42 pesticides in dates. Acidified acetonitrile and citrate buffer salts were used to extract re-hydrated samples. Acceptable validation performances were achieved, i.e., recovery range of 70−120 % and RSD values ≤20 % for 42 analytes at three different concentrations,100, 50 and 10 μg kg-1. This method was used to analyse 200 date fruit samples (v. Sukkari) collected from different large markets in the Al-Qassim region in Saudi Arabia. Pesticide residues were detected in 36 (18 %) of the date fruits samples, and 15 samples (7.5 %) exceeded the maximum residue levels. The ruggedness test results showed that this method is robust and suitable for the determination of pesticide residues in dates. Additionally, the results showed the monitored dates did not have a health impact on consumers in Saudi Arabia during the study period.

Abstract

A sensitive, simple and rapid QuEChERS extraction method and liquid chromatography equipped with triple quadrupole mass spectrometry (LC-MS/MS) were used to evaluate 42 pesticides in dates. Acidified acetonitrile and citrate buffer salts were used to extract re-hydrated samples. Acceptable validation performances were achieved, i.e., recovery range of 70−120 % and RSD values ≤20 % for 42 analytes at three different concentrations,100, 50 and 10 μg kg-1. This method was used to analyse 200 date fruit samples (v. Sukkari) collected from different large markets in the Al-Qassim region in Saudi Arabia. Pesticide residues were detected in 36 (18 %) of the date fruits samples, and 15 samples (7.5 %) exceeded the maximum residue levels. The ruggedness test results showed that this method is robust and suitable for the determination of pesticide residues in dates. Additionally, the results showed the monitored dates did not have a health impact on consumers in Saudi Arabia during the study period.

Chromatographic methods in HIV medicine: Application to therapeutic drug monitoring

HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC-UV and LC-MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid–liquid extraction or solid-phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C18). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co-infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.

Abstract

HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC-UV and LC-MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid–liquid extraction or solid-phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C18). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co-infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.

Determination of isoorientin levels in rat plasma after oral administration of Vaccinum bracteatum Thunb. methanol extract by high-performance liquid chromatography-tandem mass spectrometry

A simple, sensitive and rapid liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination of plasma isoorientin levels in rats. After simple protein precipitation using methanol, chromatographic analysis was performed using a Synergi 4μ polar-RP 80A column (150 × 2.0 mm, 4μm) under isocratic conditions and a mobile phase consisting of 0.1% formic acid in water and methanol (80:20, v/v) at a flow rate of 0.2 mL/min. In positive electrospray ionization mode, the protonated precursor and product ion transitions of isoorientin (m/z 449.0 299.1) and of puerarin (the internal standard; m/z 417.1 297.1) were acquired by multiple reaction monitoring. Calibration curves obtained for plasma showed good linearity over the concentration range 1–1000 ng/mL. The lower limit of quantification was 1 ng/mL. Intra- and inter-day precisions were within 8.8% relative standard deviation. Accuracies ranged from 92.1 and 109.7%. The isoorientin stability in rat plasma under typical handling/storage conditions also found to be acceptable. The developed method was applied successfully to a pharmacokinetic study of isoorientin orally administered as the methanol extract of Vaccinium bracteatum Thunb. or administered as pure isoorientin.

Abstract

A simple, sensitive and rapid liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination of plasma isoorientin levels in rats. After simple protein precipitation using methanol, chromatographic analysis was performed using a Synergi 4μ polar-RP 80A column (150 × 2.0 mm, 4μm) under isocratic conditions and a mobile phase consisting of 0.1% formic acid in water and methanol (80:20, v/v) at a flow rate of 0.2 mL/min. In positive electrospray ionization mode, the protonated precursor and product ion transitions of isoorientin (m/z 449.0 [RIGHTWARDS ARROW] 299.1) and of puerarin (the internal standard; m/z 417.1 [RIGHTWARDS ARROW] 297.1) were acquired by multiple reaction monitoring. Calibration curves obtained for plasma showed good linearity over the concentration range 1–1000 ng/mL. The lower limit of quantification was 1 ng/mL. Intra- and inter-day precisions were within 8.8% relative standard deviation. Accuracies ranged from 92.1 and 109.7%. The isoorientin stability in rat plasma under typical handling/storage conditions also found to be acceptable. The developed method was applied successfully to a pharmacokinetic study of isoorientin orally administered as the methanol extract of Vaccinium bracteatum Thunb. or administered as pure isoorientin.

Identification of cytochrome P450 isoforms involved in the metabolism of artocarpin and assessment of its drug–drug interaction

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti-inflammation and anticancer activities. In this study, we utilized recombinant human UDP-glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 μm, and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 μm, respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin-containing herbs.

Abstract

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti-inflammation and anticancer activities. In this study, we utilized recombinant human UDP-glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 μm, and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 μm, respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin-containing herbs.

Development and validation of LC-MS/MS method for simultaneous determination of sofosbuvir and daclatasvir in human Plasma: Application to pharmacokinetic study

The first simple and highly sensitive Liquid chromatography–tandem mass spectrometry (LC–MS/MS) bioanalytical method was developed and fully validated for the simultaneous determination of newly discovered antiviral drugs namely, Sofosbuvir (SOF) and Daclatasvir (DAC) in human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid-liquid extraction technique with methyl tert-butyl ether. The chromatographic separation was carried out using ZorbaxSB-C18 column (4.6 x 50 mm,5μm) and 5 mM ammonium formate buffer (pH 3.5): acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL min-1. The quantitation was performed on API4500 triple quadrupole tandem mass spectrometer with positive electrospray ionization interface in multiple reaction monitoring (MRM) mode. Validation was applied according to FDA guidelines for bio-analytical methodswith respect to linearity, precision, accuracy, selectivity, carry-over, stability and dilution integrity. Linearity was obtained over a concentration range of 0.3-3000 and 3-3000 ng mL-1 for SOF and DAC; respectively by applying weighted least-squares linear regression method (1/x2). The proposed method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug monitoring (TDM) of patients undergoing dual combination therapy as the latter combination proved more efficacious and powerful tool for the complete treatment of hepatitis C (HCV) genotype 3 within 16 weeks. The suggested method has been applied successfully to pharmacokinetic studies (PK) with excellent assay ruggedness and reproducibility.

Abstract

The first simple and highly sensitive Liquid chromatography–tandem mass spectrometry (LC–MS/MS) bioanalytical method was developed and fully validated for the simultaneous determination of newly discovered antiviral drugs namely, Sofosbuvir (SOF) and Daclatasvir (DAC) in human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid-liquid extraction technique with methyl tert-butyl ether. The chromatographic separation was carried out using ZorbaxSB-C18 column (4.6 x 50 mm,5μm) and 5 mM ammonium formate buffer (pH 3.5): acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL min-1. The quantitation was performed on API4500 triple quadrupole tandem mass spectrometer with positive electrospray ionization interface in multiple reaction monitoring (MRM) mode. Validation was applied according to FDA guidelines for bio-analytical methodswith respect to linearity, precision, accuracy, selectivity, carry-over, stability and dilution integrity. Linearity was obtained over a concentration range of 0.3-3000 and 3-3000 ng mL-1 for SOF and DAC; respectively by applying weighted least-squares linear regression method (1/x2). The proposed method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug monitoring (TDM) of patients undergoing dual combination therapy as the latter combination proved more efficacious and powerful tool for the complete treatment of hepatitis C (HCV) genotype 3 within 16 weeks. The suggested method has been applied successfully to pharmacokinetic studies (PK) with excellent assay ruggedness and reproducibility.