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 the bioavailability of TMP need to be further explored. In our study, an in vivo microdialysis sampling technique coupled with ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) 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 the acceptable ranges. The results demonstrated BO had a marked impact on the pharmacokinetic properties of TMP. After co-administered, AUC0-inf of TMP in brain and blood were significantly increased. Meanwhile, Cmax of TMP in brain was also enhanced. The AUCBrain/AUCBlood of TMP, respectively increased from 44% to 56% and 60.8% after co-administration with BO (15 and 30 mg/kg). The pharmacodynamics results showed that TMP co-administration with BO enhanced the CBF during the period of ischemia and reduced the infarct volume. Overall, it might be an effective way to treat stroke by using TMP co-administration 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 the bioavailability of TMP need to be further explored. In our study, an in vivo microdialysis sampling technique coupled with ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) 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 the acceptable ranges. The results demonstrated BO had a marked impact on the pharmacokinetic properties of TMP. After co-administered, AUC0-inf of TMP in brain and blood were significantly increased. Meanwhile, Cmax of TMP in brain was also enhanced. The AUCBrain/AUCBlood of TMP, respectively increased from 44% to 56% and 60.8% after co-administration with BO (15 and 30 mg/kg). The pharmacodynamics results showed that TMP co-administration with BO enhanced the CBF during the period of ischemia and reduced the infarct volume. Overall, it might be an effective way to treat stroke by using TMP co-administration 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 (Tmax 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 H/D 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 (BS) and followed by mass defect filter (MDF). NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the H/D exchange data and cLogP simulation. As a result, five phase I metabolites (M1-M5) of NQ were firstly characterized. Two metabolic pathways were involved, which included (hydroxylation and N-oxidation). This study demonstrated 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 (Tmax 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 H/D 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 (BS) and followed by mass defect filter (MDF). NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the H/D exchange data and cLogP simulation. As a result, five phase I metabolites (M1-M5) of NQ were firstly characterized. Two metabolic pathways were involved, which included (hydroxylation and N-oxidation). This study demonstrated 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

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 (ranged 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 x 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/mole). 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. Besides, this work will be highly useful in preparative chromatography where the separation is the major problem at large scale. Moreover, developed LC-MS-QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample due to the low value of detection limits.

Abstract

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 (ranged 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 x 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/mole). 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. Besides, this work will be highly useful in preparative chromatography where the separation is the major problem at large scale. Moreover, developed LC-MS-QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample due 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 (3.0×50mm, 1.7μm) 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 ranges acceptable. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10mg/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 (3.0×50mm, 1.7μm) 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 ranges acceptable. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10mg/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.

HPLC-MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute-and-shoot sample preparation methods, respectively

A high-performance liquid chromatography tandem–mass spectrometry (HPLC-MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP-Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water–1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute-and-shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, cyanidin-3-xyloside and quercetin-3-galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.

Abstract

A high-performance liquid chromatography tandem–mass spectrometry (HPLC-MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP-Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water–1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute-and-shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, cyanidin-3-xyloside and quercetin-3-galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.

The issue of HPLC determination of endogenous lipoic acid in human plasma

Lipoic acid (LA) is used extensively as a therapeutic agent for the treatment of various diseases. Many methods have been reported for the determination of LA plasma levels and its metabolites after its supplementation, but available information concerning endogenous plasma levels is still rare. Studies, which directly focused on determining the endogenous plasma levels, provided highly controversial results, less than 4.9 nmol/L or 143.7 – 197.0 nmol/L.
The main aim of this study was to verify the levels of free LA in the plasma of 40 individuals (17 women, 23 men). This group was non-supplemented with LA and met the conditions for incorporation into the blood donors register. We measured the levels of LA using an HPLC method with very sensitive coulometric detection after previous sample preparation including deproteination and solid phase extraction with phenyl cartridge.
Our limit of detection was 1.85 nmol/L and was better than the LODs in studies, which directly focused on determining the endogenous plasma levels of LA, 2.4 nmo/L and 4.9 nmol/L respectively. However, the levels of free LA in the plasma of non-supplemented voluntary blood donors were not detectable in all cases. The presented results of our study show that endogenous concentrations of LA are less than 1.85 nmol/L.

Abstract

Lipoic acid (LA) is used extensively as a therapeutic agent for the treatment of various diseases. Many methods have been reported for the determination of LA plasma levels and its metabolites after its supplementation, but available information concerning endogenous plasma levels is still rare. Studies, which directly focused on determining the endogenous plasma levels, provided highly controversial results, less than 4.9 nmol/L or 143.7 – 197.0 nmol/L.

The main aim of this study was to verify the levels of free LA in the plasma of 40 individuals (17 women, 23 men). This group was non-supplemented with LA and met the conditions for incorporation into the blood donors register. We measured the levels of LA using an HPLC method with very sensitive coulometric detection after previous sample preparation including deproteination and solid phase extraction with phenyl cartridge.

Our limit of detection was 1.85 nmol/L and was better than the LODs in studies, which directly focused on determining the endogenous plasma levels of LA, 2.4 nmo/L and 4.9 nmol/L respectively. However, the levels of free LA in the plasma of non-supplemented voluntary blood donors were not detectable in all cases. The presented results of our study show that endogenous concentrations of LA are less than 1.85 nmol/L.

A fast and accurate isotope dilution GC-IT-MS/MS method for determination of eugenol in different tissues of fish: application to a depletion study in mandarin fish

An accurate, rapid and effective method was established for determination of eugenol in plasma, muscle, skin, liver, kidney and gill of fish using gas chromatography-ion trap tandem mass spectrometry (GC-IT-MS/MS). Samples of muscle, skin, liver, kidney and gill were prepared using the modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, and plasma sample was prepared by a liquid-liquid extraction procedure. Eugenol was monitored in less than 7 min using an electron-ionization source (EI) in MS/MS mode and quantified by internal standard (IS) of eugenol-d3. The limit of detection (LOD) was 5.0 μg/kg, and the limit of quantification (LOQ) was 10.0 μg/kg. The calibration curve was linear in the range of 5–1000 μg/L (R2 = 0.9996). Intra- and inter- precisions of eugenol expressed as an RSD within 9.74%, and the accuracy exhibited an RE ranged from -2.20% and 8.89%. The developed method was successfully used to study the elimination regularity of eugenol in mandarin fish.

Abstract

An accurate, rapid and effective method was established for determination of eugenol in plasma, muscle, skin, liver, kidney and gill of fish using gas chromatography-ion trap tandem mass spectrometry (GC-IT-MS/MS). Samples of muscle, skin, liver, kidney and gill were prepared using the modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, and plasma sample was prepared by a liquid-liquid extraction procedure. Eugenol was monitored in less than 7 min using an electron-ionization source (EI) in MS/MS mode and quantified by internal standard (IS) of eugenol-d3. The limit of detection (LOD) was 5.0 μg/kg, and the limit of quantification (LOQ) was 10.0 μg/kg. The calibration curve was linear in the range of 5–1000 μg/L (R2 = 0.9996). Intra- and inter- precisions of eugenol expressed as an RSD within 9.74%, and the accuracy exhibited an RE ranged from -2.20% and 8.89%. The developed method was successfully used to study the elimination regularity of eugenol in mandarin fish.

Analysis and measurement of serotonin

Serotonin, also known as 5-hydroxytryptamine, is an important signaling molecule in the central and peripheral nervous systems of humans. Acting through several receptor types, it helps regulate the normal functioning of the gastrointestinal tract, cardiovascular system and brain. Serotonin signaling has also been implicated in the etiology of several diseases, including depression, anxiety disorders, hypertension and irritable bowel syndrome. The present review focuses on the chemical analysis of serotonin in biological fluids and biomatrices and traces the development and application of early methods based on UV absorbance or fluorescence to more widely used current methods such as high-performance liquid chromatography coupled to mass spectrometry. A brief summary of the biochemistry, metabolism and physiological roles of serotonin is also presented.

Abstract

Serotonin, also known as 5-hydroxytryptamine, is an important signaling molecule in the central and peripheral nervous systems of humans. Acting through several receptor types, it helps regulate the normal functioning of the gastrointestinal tract, cardiovascular system and brain. Serotonin signaling has also been implicated in the etiology of several diseases, including depression, anxiety disorders, hypertension and irritable bowel syndrome. The present review focuses on the chemical analysis of serotonin in biological fluids and biomatrices and traces the development and application of early methods based on UV absorbance or fluorescence to more widely used current methods such as high-performance liquid chromatography coupled to mass spectrometry. A brief summary of the biochemistry, metabolism and physiological roles of serotonin is also presented.