UPLC–MS/MS for the determination of azilsartan in beagle dog plasma and its application in a pharmacokinetics study

The purpose of the study is to develop an ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) to determinate the concentration of azilsartan in the dog plasma. After precipitated by methanol, the plasma sample containing azilsartan and diazepam (internal standard, IS) was determined by UPLC–MS/MS. The mobile phase consisted of acetonitrile-water was pumped at a flow rate of 0.3 ml/min in gradient elution. Kinetex 2.6 μ XB-C18 column (50 × 2.1 mm, 100 Å; Phenomenex, USA) were used for LC separations. The column temperature was 30 °C and the injection volume was 5 μl. The electrospray ionization (ESI) and multiple reaction monitoring (MRM) were applied at the transitions of m/z 457 → 279 (azilsartan) and m/z 285 → 193 (diazepam), respectively. The developed method was identified a good linearity over a concentration range of 2.5–5000 ng/ml. The lower limit of quantitation (LLOQ) was 2.5 ng/ml. The intra-day and inter-day precision (relative standard deviation, RSD%) were less than 10% and accuracy (relative error, RE %) was less than 5% at three quality control levels. The extraction recovery of azilsartan at three quality control levels were 82.41 ± 0.68%, 98.66 ± 11.00%, 102.43 ± 0.82%. And the recovery for IS (100 ng/ml) was 91.75 ± 0.54%. A validated UPLC–MS/MS method was firstly developed for the quantification of azilsartan in dog plasma and it was applied to the pharmacokinetics study.     

Boron’s journey: advances in the study and application of pharmacokinetics

Introduction: Boron-containing compounds (BCCs) are attractive chemical entities in drug development. Some of these compounds have been used in the treatment of human disease, and studies on their pharmacodynamics suggest that they employ multiple forms of activity. However, less is known about the pharmacokinetic profile of these molecules.

Areas covered: The herein compiled reported data is presented in accordance with the classical ‘ADME’ system for identifying the scope of BCCs in the respective fields. Our analysis suggests that these compounds have several distinct ways to move within the human body, and that the specific structural features of each molecule account for its distinct pharmacokinetic profile. These insights should be useful for designing BCCs with a desired effect.

Expert opinion: Increasing knowledge about the pharmacokinetics of BCCs is providing a broader understanding about the design of new release systems and potential drugs, as well as probable protein transporters that could be related to key roles in physiological processes. These transporters may be involved in sodium transport, hormone release and regulation of the cell cycle. The shared features among groups of BCCs are being identified in order to apply these insights to the design of advantageous compounds.     

Development and validation of a highly sensitive LC–MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study

A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC–MS/MS). A minimal volume of human plasma sample (20 μL) was prepared by simple deproteinization with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1 % formic acid at a flow rate of 0.3 mL/min over a total run time of 2.0 min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22 → 242.17 for acacetin and m/z 277.59 → 175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1–500 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9 %, and the intra- and inter-day accuracy ranged from 96.8 to 108 %. Mean recovery of acacetin in human plasma was within the range of 91.5–95.6 %. This validated LC–MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.     

Determination of bencycloquidium bromide in human urine using weak cation-exchange solid-phase extraction and LC–ESI-MS: Method validation and application to kinetic study of urinary excretion

A sensitive LC–ESI-MS method has been developed and validated for the determination of bencycloquidium bromide (BCQB) in human urine samples. The method utilized a solid-phase extraction (SPE) procedure, choosing carboxy propyl phase (CBA) as the extracting sorbent for purification of BCQB, with better baseline and higher selectivity achieved. Sample preparation by this method yielded very good and consistent mean recovery of above 94.5%. Another major benefit of the present method was the high detectability, with a lower limit of quantification (LLOQ) of 0.02 ng/ml. The developed method was successfully applied to determine BCQB in human urine, and was proved to be suitable for use in Phase I clinical pharmacokinetic study of BCQB.     

An LC–MS/MS method for the simultaneous determination of chlorogenic acid,forsythiaside A and baicalin in rat plasma and its application to pharmacokinetic study of Shuang-huang-lian in rats

An LC–MS/MS method was developed for the simultaneous determination of chlorogenic acid, forsythiaside A and baicalin, three major ingredients in Shuang-huang-lian preparations, in rat plasma. Following extraction by methanol–ethyl acetate–trifluoroacetic acid (49:49:2, v/v/v), the extracted analytes were separated on a reverse phase C12 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The limits of quantification were between 1.0 and 2.1 ng/mL, the precision was <7% and the accuracy was between 94% and 107%. The validated method was applied to a comparative pharmacokinetic study in rats after administration of Shuang-huang-lian solutions via intravenous, peroral or intratracheal routes. The results showed that the three chemical markers were more rapidly and thoroughly absorbed following pulmonary delivery as compared with peroral administration.     

Quantitative determination of pimozide in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study

A simple, sensitive and specific LC–ESI/MS method was developed for the determination of pimozide in human plasma. Pimozide and cinnarizine (internal standard) were isolated from plasma samples by liquid–liquid extraction. The chromatographic separation was accomplished on a Thermo Hypersil-HyPURITY C18 reversed-phase column (150 mm × 2.1 mm, i.d., 5 μm) with the mobile phase consisting of 5 mM ammonium acetate (pH 3.5, adjusted with acetic acid)–methanol–acetonitrile (39:5:56, v/v/v). The lower limit of quantification was 0.02 ng/mL, and the assay exhibited a linear range of 0.025–12.800 ng/mL. The established method has been successfully applied to a bioequivalence study of 2 pimozide formulations in 32 healthy male Chinese volunteers.     

Development of a method for determination of osmotic pump controlled-release tablets of lorcaserin hydrochloride in beagle dog plasma and its application to pharmacokinetic study北大核心CSCD

Aim To develop an UPLC-MS/MS method to determine the concentration of lorcaserin hydrochloride in beagle plasma, and study the pharmacokinetics of osmotic pump controlled-release tablets of lorcaserin hydrochloride. Methods A randomized crossover design was used, carbamazepine as the internal standard(IS), and plasma protein precipitation with acetonitrile. The chromatographic was Phenomenex Polar C18 column(100 mm×2. 1 mm, 3 μm), and acetonitrile - water(containing 10 mmol·L-1 ammonium acetate and 0.1% formic acid)(40:60, V/V)was mobile phase. Multiple reaction monitoring mode and electrospray positive ionization were used to detect lorcaserin hydrochloride. The MS/MS ion transitions were monitored at m/z 196.2→129.2 for lorcaserin hydrochloride and m/z 237→194.1 for carbamazepine, respectively. Results The linear range was 1 to 500 μg·L-1(r=0.999 2), the extraction recovery rate ranged from 87.70% to 89.70%, the precision RSD was 9.7%. The accuracy and matrix effect met the requirements, and the stability of lorcaserin hydrochloride was good in -20 ℃ refrigerator for 45 d, repeated freezing and thawing for three times, placed at room temperature for 24 h, and the disposed samples placed in automatsampler for 6 h were stable. The main pharmacokinetic parameters of the controlled-release tablet and immediate-release tablet were as follows:Tmax was(8.00±1.27)h and(1.00±0.13)h, Cmax was(70.56±3.73)μg·L-1 and(176.33±16.73)μg·L-1, and AUC0-t was(966.33±7.56)μg·h·L-1 and(973.05±69.09)μg·h·L-1, respectively. Conclusions The established UPLC-MS/MS method can be used to study the pharmacokinetics of lorcaserin hydrochloride in the plasma of beagle dogs, and osmotic pump controlled-release tablets has sustained release effect. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

GC-MS method for the simultaneous determination of β-blockers, flavonoids, isoflavones and their metabolites in human urine

A sensitive and selective method based on gas chromatography hyphenated to mass spectrometry (GC-MS) for the screening of 23 different compounds including β-blockers, flavonoids, isoflavones and metabolites in human urine sample was developed and validated. The present paper reports, for the first time, the method for the simultaneous determination of β-blockers, isoflavones, flavonoids and metabolites in human urine samples. When flavonoids are ingested in combination with drugs that have a narrow therapeutic range, interactions between flavonoids and drugs should be investigated.Substances of interest were extracted from urine samples by solid-phase extraction (SPE) employing a mixture of tert-butyl methyl ether:methanol:formic acid (4.5:4.5:1; v/v/v) as a mobile phase and Oasis HLB (Waters) as a stationary phase. Before extraction, urine samples were incubated with β-glucuronidase/sulfatase in order to achieve enzymatic hydrolysis. Before GC-MS analysis the analytes had to be derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) into their trimethylsilyl derivatives by incubating for 60 min at 60 °C. Statistical central composite design and response surface analysis were used to optimize the derivatization reagent. These multivariate procedures were efficient in determining the optimal separation condition, using peak areas as responses.The calibration curves were indicative of high linearity (r² ≥ 0.9992) in the range of interest for each analyte. LODs (S/N = 3) ranged between 0.6 and 9.7 ng/ml. Intra-day and inter-day precision (CV, %) was less than 4.96%, accuracy between 0.01 and 4.98% and recovery was found in the range from 70.20 to 99.55%.The developed method can be applied to the routine determination of examined compounds’ concentrations in human urine. Moreover the method is suitable for detecting pharmaceutical compounds containing β-blockers, isoflavones and flavonoids in urine after administration to humans.     

Reverse transcription polymerase chain reaction cDNA cloning and its application in the molecular toxicology

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Pharmacokinetic properties of γ-hydroxybutyrate (GHB) in whole blood, serum, and urine

Over the last 10-15 years, γ-hydroxybutyrate (GHB) and γ-butyrolactone have become increasingly popular "club drugs", but they have also gained attention as potential agents of drug-facilitated sexual assault (DFSA). Several studies have attempted to characterize GHB's pharmacokinetic properties in humans, and the aim of this paper is to build on this research with an emphasis on DFSA cases. A 25 mg/kg dose of GHB was given to 12 GHB-na?ve volunteers (6 men and 6 women). Urine and blood samples (serum and whole blood) were collected and analyzed by gas chromatography-mass spectrometry following liquid-liquid extraction. The urinary T(max) was 1 h in 11 volunteers with a mean C(max) of 67.6 mg/L (32.6-161.3 mg/L). Urinary concentrations rapidly decreased to < 10 mg/L (interpretive limit) for 11 volunteers after just 4 h. Data derived from whole blood (mean C(max) = 48.0 mg/L, T(max) = 24.6 min) closely matched that from serum (mean C(max) = 59.4 mg/L, T(max) = 23.3 min), suggesting GHB is distributed into erythrocytes. All 12 volunteers had GHB concentrations of less than 5 mg/L in both whole blood and serum after 3 h. Results verify the rapid elimination of GHB and the limited retrospective power of a concentration-based approach to prove GHB administration in blood and urine and confirm that, in DFSA cases, samples should be collected as soon as possible.     

Immunoassay screening of diphenhydramine (Benadryl®) in urine and blood using a newly developed assay

Diphenhydramine (DPH) is a common over the counter antihistamine that produces drowsiness and has the potential to cause driving under the influence of drugs-related accidents. To date there are no commercially available immunoassay screening kits for its detection in biological fluids such as urine and/or blood. We describe a newly developed enzyme-linked immunosorbent assay (ELISA) screen and report on its utility in the analysis of authentic specimens taken from volunteers. The assay is specific for detection of DPH and does not detect closely related antihistamines like brompheniramine, chlorpheniramine, and doxylamine. There is a varying amount of cross-reactivity seen with certain tricyclic compounds, due to similarities in side chain structure with DPH. Intra- and interday precision of the assay were determined to be less than 10%. The assay is highly sensitive and has a working range from 1 to 500 ng/mL for urine and 1 to 250 ng/mL for blood. The assay was further validated with authentic urine and blood specimens obtained from volunteers and coroner's laboratories.     

Simultaneous determination of γ-Hydroxybutyrate (GHB) and its analogues (GBL, 1.4-BD, GVL) in whole blood and urine by liquid chromatography coupled to tandem mass spectrometry

A simple liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous identification and quantification of γ-hydroxybutyrate (GHB), γ-butyrolactone (GBL), 1.4-butanediol (1.4-BD), and γ-valerolactone (GVL) in whole blood from forensic cases. The sample preparation of whole blood involved protein precipitation by acidic methanol. Urine samples were diluted and evaluated in relation to a control at the cutoff concentration. Hexadeutero GHB (GHB-d(6)) was used as the internal standard. Separation was achieved by reversed-phase chromatography, and detection was by MS-MS in MRM mode. The linear range for all compounds was from 1.0 to 100 mg/kg in whole blood with a limit of quantification of about 1 mg/kg. The method was validated with regards to selectivity, recovery, accuracy and precision, and stability. The method is currently applied to investigations on suspected drug-facilitated sexual assaults, driving under the influence of drugs, and general intoxication with these substances.     

Detection and quantitation of β-blockers in plasma and urine

β-blockers are a class of antihypertensive drugs that are used for the management of cardiac arrhythmias, cardioprotection after myocardial infarction (heart attack) and hypertension. They have revolutionized the medical management of angina pectoris and are recommended as first-line agents by national and international guidelines. Although β-blockers are still the cornerstone for the treatment of heart failure, some of the drugs in this category are prohibited in several sports requiring vehicle control and bodily movements as they reduce heart rate and tremors, and improve performance. As a result, urine analysis of β-blockers is mandatory in doping control and toxicological screening. The determination of plasma levels of β-blockers helps to ensure noncompliance in patients with persistent hypertonia to confirm the diagnosis of β-blocker poisoning and for therapeutic drug monitoring. This review provides a comprehensive account of various analytical methods developed for detection and quantitation of β-blockers in plasma and urine.     

Verifying the validity of creatinine measurement in low-concentrated urine spot samples—Photospectrometry versus liquid chromatography–tandem mass spectrometry

One of the major challenges of testing drugs of abuse is the detection of highly diluted urine samples. The ingestion of a large amount of fluid can considerably reduce the concentration of substances, possibly resulting in inaccurate drug testing. For detection, determination of urinary creatinine is a widely established procedure. In this study, results from the most popular methods, including photospectrometry (Jaffe) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), have been compared regarding 327 urine abstinence control samples. Because samples with creatinine concentrations close to the cutoff of 20 mg/dL are of particular interest, only samples below 50 mg/dL were considered. Results revealed a close correlation of creatinine concentrations by both analytical methods with an R² value of 0.9005. A mean concentration difference of 3.30 ± 3.45 mg/dL was observed, indicating a moderate underestimation by the Jaffe reaction. Graphical analyses showed high accordance between both methods with only a few outliers. Due to easy handling and for economic reasons, the spectrometric method is often preferred over LC–MS/MS. For urine samples with creatinine concentrations close to the cutoff, confirmation through a second method should be performed to avoid a possible disadvantage or even severe consequences for the respective individual. It is recommended that each laboratory establishes a reliable verification method.     

Development and validation of an EI–GC–MS method for the determination of benzodiazepine drugs and their metabolites in blood: Applications in clinical and forensic toxicology

Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid–liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.     

Validated LC–MS/MS method for the determination of sarpogrelate in human plasma: Application to a pharmacokinetic and bioequivalence study in Chinese volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the determination of sarpogrelate in human plasma. One-step protein precipitation with acetonitrile was used to extract the analytes from the plasma. Sarpogrelate and tramadol (internal standard, I.S.) were separated on a Venusil MP-C₁₈ column within 1.7 min, using acetonitrile:ammonium acetate (10 mM, pH 6.8) (55:45, v/v) as mobile phase at a flow rate of 1.2 mL/min with an approximately 1:1 split entering the mass spectrometer. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of sarpogrelate at m/z 430.3 → 135.3 and of I.S. at m/z 264.1 → 58.0. The assay was validated over the concentration range of 1–1000 ng/mL with a lower limit of quantitation (LLOQ) of 1 ng/mL using 50 μL of plasma. The intra- and inter-day precision (relative standard deviation, R.S.D.) were ≤6.4% and ≤5.4%, respectively, with accuracy (relative error, R.E.) in the range 0.5–3.6%. The method was successfully applied to a pharmacokinetic and bioequivalence study enrolling 22 Chinese volunteers administered sarpogrelate tablets.     

Bioavailability assessment: Methods to estimate total area (AUC 0-∞) and total amount excreted (A e ∞ ) and importance of blood and urine sampling scheme with application to digoxin

Five methods are compared to estimate the total area under the digoxin plasma or serum concentrationtime curve (AUC0-) after a single dose of drug. To obtain accurate estimates of AUC0-, data required are concentrations at a sufficient number of sampling times to define adequately the concentration-time curve prior to the log-linear phase, and at least three, but preferably four or more equally spaced points in the terminal loglinear phase. One method (designated Method I) requires a digital computer; another (Method III) is the classical method (these two methods do not require equally spaced points in the loglinear phase). Method IIA is the accelerated convergence method of Amidon et al.; Methods IIB and IIC are modifications of this method, but incorporate usual and orthogonal least squares, respectively, which make them more accurate with real (noisy) data. Methods I and IICgave very comparable estimates of AUC0-. Results indicate that digoxin administered orally in aqueous solution was completely (100%) absorbed when bioavailability estimates were based on oral and intravenous AUC0- estimates and the actual doses, whereas formerly only about 80% absorption was reported, based on areas, under plasma concentration curves which were truncated at 96 hr. It is shown that the sampling scheme of blood can produce biased apparent bioavailability estimates when areas under truncated curves are employed, but an appropriate sampling scheme and application of method IIyield accurate bioavailability estimates. This is important particularly in those bioavailability studies where one is attempting to determine the appropriate label dose for a new fastrelease digoxin preparation relative to the label dose and bioavailability of currently marketed tablets. It is shown that the magnitudes and variability of apparent elimination rate constants and halflives of digoxin, estimated from urinary excretion data by the ^– method, depend on which value of A _e is used. The formerly reported greater interindividual variability of AUC data compared to At data for digoxin is explained in that the AUCs, but not the A_e,'s, involve the renal clearance, which exhibits considerable inter- and intraindividual variation.