Ultra-high Pressure Liquid Chromatography-tandem Mass Research Articles

Determination of regulated perfluoroalkyl substances (PFAS) in drinking water according to Directive 2020/2184/EU.

Perfluoroalkyl substances (PFAS) are chemical compounds that have been widely used in industry and manufacture. Occurrence, together with persistence and recent toxicological effects data, has promoted the regulation of 20 PFAS (carboxylic and sulfonic) acids in drinking water through the recent Directive 2020/2184/EU. This Regulation included PFAS with different carbon chain lengths (from C4 to C13) and limited the total PFAS concentration (as sum) to a maximum of 0.1µg/L, for which law-enforcement analytical methods are required. In this work, three different methodologies have been developed and evaluated as regards their performance to determine those 20 PFAS in tap and bottled water, based on on-line and off-line solid-phase extraction (SPE) and direct injection. In all cases, ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used as a determination technique. Off-line SPE with Oasis Weak Anion Exchange (WAX) cartridges provided the best results in terms of limits of quantification (LOQ ≤ 0.3ng/L) and accuracy (R ≥ 70%) in drinking water samples. On-line SPE and direct injection presented some drawbacks such as background contamination problems and lower accuracies for the least polar compounds. This off-line SPE methodology was then applied to the analysis of 46 drinking water samples (11 commercial bottled samples, 23 Spanish and 12 international tap water samples). Ten PFAS were quantified in such samples at concentrations and detection frequencies ranging from 0.1 to 20.1ng/L and 2 to 91%, respectively. However, the sum concentration did not surpass the established limit in any sample.

Rapid analysis of N-nitrosamines in urine using ultra high-pressure liquid chromatography-mass spectrometry.

N-Nitrosamines, carcinogenic compounds present in dietary and environmental sources and formed endogenously, are believed to be linked with the presence of nitrate and nitrite, both within dietary sources and after intake. To fully evaluate this potential threat to human health, an accurate analytical method to measure N-nitrosamines in biological matrices is necessary. We report a simple, fast, selective mass spectrometry method to detect N-nitrosamines in human urine. Analysis of seven N-nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosopiperdine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosodi-N-propylamine (NDPA) and N-nitrosodi-N-butylamine (NDBA) in urine was quantitated using Ultra High-Pressure Liquid Chromatography-tandem Mass spectrometry (UHPLC-MS/MS). A Sorbent supported Liquid Extraction (SLE) method was employed to extract N-nitrosamines from 24 hour collected human urine samples. The percent recovery varied between 74.3 to 110 and the limit of detection and limit of quantification ranged from 0.1 to 0.85 ng mL-1 and 0.22 to 2.06 ng mL-1 respectively. Precision for inter-day and intra-day assay yielded a % coefficient of variation between 4-10% for all measured compounds in urine. Linear regression analysis of calibration curves for N-nitrosamines measured in urine in the concentration range 0.4-12.8 ng mL-1 gave correlation coefficients, R2 0.9874-0.9962. Urinary excretion of N-nitrosamines measured in ten healthy subjects resulted in detection of most of the N-nitrosamines including NDMA, NDEA, NPYR, NDPA and NDBA by this method.

Analyzing Per- and Polyfluoroalkyl Substances (PFAS) in Foods

Environmental contaminants can affect human and animal health and are thus an issue of concern. Anthony Provatas is a Research Professor and the Organics Laboratory Manager at the University of Connecticut in the Center for Environmental Sciences and Engineering (CESE) of the Institute of the Environment; he is also Program Chair and Board Member of the Eastern Analytical Symposium (EAS). His research has been focused on the effects of contaminants on human health. We recently spoke to him about his work using ultrahigh-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS), and UHPLC–quadrupole–time-of-flight mass spectrometry (UHPLC– QTOF-MS) to analyze the presence of per- and polyfluoroalkyl substances (PFAS) in the environment, particularly in foods.

Persistent toxicity and dissipation dynamics of afidopyropen against the green peach aphid Myzus persicae (Sulzer) in cabbage and chili

The green peach aphid, Myzus persicae (Sulzer), is a significant global pest in horticultural and field crops. Afidopyropen is a novel systemic insecticide with high efficacy against sucking pests, and it is suitable for the management of M. persicae. However, the persistent toxicity and dissipation dynamics of afidopyropen in vegetables remain unknown. In this study, we determined the residual activity and dissipation dynamics of afidopyropen against M. persicae on cabbage and chili. The data showed that the toxicity of afidopyropen against M. persicae lasted more than 30 days; the corrected mortality was greater than 80% 10 days after application and was 50–60% 30 days post-application. The afidopyropen residues on cabbage and chili plants were quantified using ultrahigh-pressure liquid chromatography–tandem mass spectrometry. The dissipation half-lives of afidopyropen on cabbage and chili plants ranged from 1.45 to 2.34 days and 3.98–5.98 days at different recommended dosages, respectively. Our findings provide valuable data for the maximum residue limits of afidopyropen on vegetables and will help growers determine the frequency and timing of its application on cabbage and chili.

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Early detection of lipid oxidation in infant milk formula by measuring free oxylipins-Comparison with hydroperoxide value and thiobarbituric acid reactive substance methods.

Infant milk formula was used as a model food to compare the sensitivity of thiobarbituric acid reactive substances (TBARS) and hydroperoxide methods to UPLC-MS/MS oxylipin analysis for detecting early lipid oxidation. Two different infant milk formulas were tested during 21 days of storage at 4°C. Formulas 1 and 2 contained canola oil and canola oil + 1% docosahexaenoic acid ethyl ester, respectively. Formulas were sampled up to 21 days of storage. Formula 2 had higher peroxide values than Formula 1 across all time points. However, no significant differences over time in TBARS and peroxide values in either formula were observed. Several oxylipins increased in both formulas starting on day 7 (linoleic acid and alpha-linolenic acid-derived oxylipins in Formula 1 and DHA-derived oxylipins in Formula 2). These results indicate that free oxylipins are effective in detecting early lipid oxidation and distinguishing between formulations containing different fatty acids. PRACTICAL APPLICATION: We have recently shown that primary oxidation products known as oxylipins can be measured in their free form by ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect early lipid oxidation. However, a head-to-head comparison of the sensitivity of this approach to conventional spectrophotometric methods has not been evaluated. Our results indicate that free oxylipin measurements are better than conventional methods in detecting early lipid oxidation in milk infant formula distinguishing between different formulations.

Survey of per- and polyfluoroalkyl substances (PFAS) in surface water collected in Pensacola, FL

As the persistence of per- and polyfluoroalkyl substances (PFAS) become a global concern, information about the occurrence and characteristics of PFAS in estuarine and marine ecosystems is poorly represented. In this study, the presence of 51 PFAS were monitored in the Pensacola Bay System (PBS), Florida, USA. Due to the presence of many potential PFAS sources in close proximity to the PBS (e.g., military bases, industries, airports and several firefighting stations), the distribution and concentration of PFAS in this estuarine environment provides insights into the fate of these complex compounds as well as the possible impacts on coastal systems. Surface water was collected and analyzed from 45 different sites via Strata-X-AW cartridge extractions and ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Recoveries for many PFAS (13/51) were >60% (mean 77 %), with relative standard deviations below 20%, except for N-methylperfluoro-1-octanesulfonamidoacetic acid (N-MeFOSAA) (22%). Of the perfluoroalkyl carboxylic acids (PFCAs), which comprised the majority of PFAS detected: perfluorooctanoic acid (PFOA) and perfluorohexanoic acid (PFHxA) were present in all samples; however, perfluoropentanoic acid (PFPeA) was the individual PFAS with the highest concentration of this group (51.9 ng.L−1, at site 81). The PFAS detected at the highest concentrations were perfluoroalkyl sulfonic acids (PFSA), with perfluorooctane sulfonic acid (PFOS) having the highest detected concentration (269 ng.L−1, at site 81). At all sites, at least eight or more PFAS were quantified. Past and current use of PFAS-containing materials and their fate in areas surrounding military bases, airports, and industries, require more in-depth monitoring efforts to better determine the need for regulation, management, and/or remediation. Here, sites located close to areas suspected of PFAS use had elevated concentrations. For example, one coastal location near an airfield had a ΣPFAS of 677 ng.L−1. Expansion from these ongoing efforts will focus on assessment of PFAS-related effects in local wildlife and evaluating the distribution of PFAS at these “hotspot” sites during large episodic weather events, a critically understudied phenomenon regarding PFAS and vulnerable coastal environments.

Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes.

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC–MS/MS (ultrahigh-pressure liquid chromatography–tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The Vmax of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the Vmax of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication–drug and drug–enzyme interactions.

A porous boron nitride nanorods-based QuEChERS analysis method for detection of five neonicotinoid pesticide residues in goji berries

To accurately determine neonicotinoid pesticide residues in goji berries, porous boron nitride nanorods (p-BNNRs) were prepared and used as a new QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) clean-up sorbent. Combined with ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), a modified QuEChERS method was developed to determine five neonicotinoid pesticide residues in goji berries. In goji berries, the p-BNNRs were shown to have a greater clean-up ability than typical clean-up materials (C18, PSA) The recoveries of the five targets ranged from 78.1 to 117.3% at three fortified levels, and the LODs ranged from 2.2 to 3.7 μg kg−1. The results indicate that this approach could be successfully used to quickly determine of the five neonicotinoid insecticide residues in goji berries for risk assessment purposes, demonstrating the applicability and suitability of p-BNNRs for the routine evaluation of neonicotinoid insecticide residues in goji berries.

Enantioselective Assay in Bovine Tissues Containing Clenbuterol by Ultrahigh Pressure Liquid Chromatography-Tandem Mass Spectrometry

Enantioselective Assay in Bovine Tissues Containing Clenbuterol by Ultrahigh Pressure Liquid Chromatography-Tandem Mass Spectrometry

Development and Validation of an UHPLC-MS/MS Assay for the Therapeutic Monitoring of Brivaracetam Plasma Concentrations in Patients with Epilepsy.

Brivaracetam is an antiepileptic drug used as an add-on therapy for partial-onset seizures in subjects aged 4 years and older. Owing to potential drug interactions and intersubject variability in plasma concentrations, therapeutic monitoring for brivaracetam may be useful. The aim of this study was to develop a simplified method for measuring brivaracetam plasma concentrations applicable to therapeutic drug monitoring in epilepsy. An ultra high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated according to current guidelines for bioanalytical methods. Sample preparation (100 µL) involved only a simple precipitation step by acetonitrile. Brivaracetam-d7 was used as internal standard. The chromatographic analysis was performed by a Synergi Fusion column using 0.1% formic acid in water/acetonitrile as a binary gradient mobile phase, at a flow rate of 0.3 mL/min. Both brivaracetam and the internal standard eluted at 1.01 minutes. This method was applied to measure trough and 1-hour postmorning dose brivaracetam plasma concentrations of 11 patients with epilepsy. The method was validated over a concentration range of 0.10-10 mcg/mL. The mean recovery was 95%. Both intra- and inter-assay imprecision and inaccuracy were <15% for all quality control samples. The lower limit of quantitation and detection was 0.10 and 0.05 mcg/mL, respectively. No interferences or carry-over was observed. Median (25%-75% quartiles) trough and 1-hour postdosing brivaracetam plasma concentrations were 0.61 mcg/mL (0.47-0.83 mcg/mL) and 1.55 mcg/mL (1.24-2.12 mcg/mL), respectively, at a median dose of 80 mg/d (50-150 mg/d). Large, up to 8-fold, intrasubject fluctuations of brivaracetam concentrations between trough and 1-hour postdosing were observed. The present assay is faster and simpler than previously published analytical reports for brivaracetam in human plasma and is suitable for therapeutic drug monitoring.

Increasing Doses of Blueberry Polyphenols Alters Colonic Metabolism and Calcium Absorption in Ovariectomized Rats.

Blueberries are rich sources of bioactive polyphenols that may provide health benefits when consumed regularly, leading to their increased marketing as dietary supplements. However, the metabolic changes associated with consuming concentrated doses of purified polyphenols, as may be present in dietary supplements, are unknown, especially when considering the colonic metabolites formed. This study aimed to evaluate the pharmacokinetics of high doses of purified blueberry polyphenols. 5-month old, ovariectomized Sprague-Dawley rats are acutely dosed with purified blueberry polyphenols (0, 75, 350, and 1000mg total polyphenols per kg body weight (bw)) and 45 Ca to measure calcium absorption. Blood and urine are collected for 48 h after dosing and phenolic metabolites measured via ultra high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The most prominent metabolites are colonically generated cinnamic and hippuric acids. Smaller amounts of other phenolic acids, flavonols, and anthocyanins are also detected. Most metabolites follow a dose-response relationship, though several show saturated absorption. Maximal metabolite concentrations are reached within 12 h for a majority of compounds measured, while some (e.g., hippuric acid) peaked up to 24 h post-dosing. Calcium absorption is significantly increased in the highest dose group (p = 0.03). These results indicate that increased doses of blueberry polyphenols induce changes in intestinal phenolic metabolism and increase calcium absorption.

Pharmacokinetic Herb‐Drug Interaction between Hibiscus sabdariffa Calyces Aqueous Extract and Captopril in Rats

Hibiscus sabdariffa L. (Malvaceae) is a traditional medicinal herb widely consumed as a beverage (“hibiscus tea”), and its global popularity is expanding due to health benefits such as blood pressure and cholesterol control. Previous studies showed that Hibiscus sabdariffa is coadministered with antihypertensives and antihyperlipidemics, thus predisposing herb-drug interactions. We investigated the pharmacokinetic interaction between H. sabdariffa L. aqueous extract and captopril, a frequently prescribed antihypertensive. In this study, chemical profile of H. sabdariffa L. aqueous extract was identified using HPLC system equipped with a DAD detector at 360 nm and 520 nm. The male Sprague Dawley rats were divided into two groups of six rats. Group I received a single dose of captopril suspension (4.5 mg/200 g body weight (BW) orally (p.o.)) while group II received H. sabdariffa L. aqueous extract (60 mg/200 g BW; p.o.) daily for two weeks prior to the same captopril dose. Multiple blood samples were collected at predetermined times after captopril administration and the plasma concentration was analyzed using ultrahigh-pressure liquid chromatography-tandem mass spectrometry. Chemical profiling of the H. sabdariffa L. aqueous extract showed that the extract contains chlorogenic acid, myricetin 3-arabinogalactoside, 5-O-caffeoylshikimic acid, quercetin 3-rutinoside, delphinidin 3-sambubioside, and cyanidin 3-sambubioside. Ingestion of the extract significantly reduced the captopril area under the curve (AUC)0−t (0.1745 (0.1254–0.2429)), AUC0−∞ (0.1734 (0.1232–0.2442))], and peak plasma concentration (0.2119 (0.1337–0.3359)) (geometric mean ratio of the coadministration group to the captopril group (90% CI)). The geometric mean ratios were falling outside the 90% CI of 0.8–1.25 bioequivalent range. Conversely, H. sabdariffa L. extract increased the apparent total body clearance (Cl/F, 0.0257 ± 0.0115 vs. 0.1418 ± 0.0338 mL/h·kg) and the apparent volume of distribution (Vd/F, 0.0541 ± 0.0226 vs. 0.3205 ± 0.0790 mL/kg). This study indicated that coadministration of H. sabdariffa L. aqueous extract could change the pharmacokinetic profile of captopril; therefore, its coadministration should be avoided.

Inkjet-Based Dispersive Liquid-Liquid Microextraction Method Coupled with UHPLC-MS/MS for the Determination of Aflatoxins in Wheat.

A novel method was developed for determination of aflatoxin B1, B2, G1, and G2 (AFB1, AFB2, AFG1, and AFG2) in wheat using inkjet-based dispersive liquid-liquid microextraction (DLLME) coupled with ultrahigh-pressure liquid chromatography-tandem mass spectrometry. A drop-on-demand jetting device was used to form a cloudy solution in traditional DLLME by injecting extraction solvent (10 μL) as ultrafine droplets (∼20 μm diameter) at high frequency into sample solution. The method was validated using wheat as a representative matrix, which was pretreated with acetonitrile/water solution. Good linearity was observed over the studied range (0.06-6 μg/kg), and the limits of quantification (0.06-0.18 μg/kg) were below the maximum level established by the European Union for cereal. Satisfactory recoveries, ranging from 83.2% to 93.0% with relative standard deviations below 4.6%, were obtained for all compounds. The method, which is convenient and reliable and has low solvent consumption, represents a new direction for the development of traditional DLLME technology.

Quantification of Lappaconitine in Mouse Blood by UPLC-MS/MS and Its Application to a Pharmacokinetic Study.

Lappaconitine is extracted from Aconitum sinomontanum Nakai, which belongs to the Ranunculaceae. Lappaconitine is as a diterpenoid alkaloid used as a nonaddictive analgesic. To assure the rational use of the drug, ultrahigh-pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was conducted to determine lappaconitine in mouse blood and its application to pharmacokinetics. In this study, khasianine was used as internet standard (IS). A UPLC BEH C18 column was used for chromatographic separation and the mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid). The flow rate of was 0.4 mL/min. Quantitative detection was performed in a multiple reaction monitoring (MRM) mode using an electrospray ionization source in positive mode. Twenty-four mice were randomly divided into four groups, three of which received 2, 4, and 8 mg/kg lappaconitine by intragastric administration, while the other group received 1 mg/kg lappaconitine by intravenous administration. After 0.0833, 0.5, 1, 1.5, 2, 3, 4, and 8 h, blood samples were collected and acetonitrile was used for protein precipitation. A linear calibration relationship (R2 = 0.9979) in the range of 0.1-500 ng/mL in mouse blood indicated good results. The lower limit of quantitation was 0.1 ng/mL and the limit of detection was 0.04 ng/mL. The intra-day and inter-day precision were below 13% and 14%, respectively. The accuracy was 90.1-107.2%, and the recovery exceeded 81.1%. The matrix effect ranged between 102.1 and 108.8%. The absolute bioavailability of lappaconitine was 2.0%. UPLC-MS/MS achieved high sensitivity, speed, and selectivity. Methodological verification indicated this method as suitable for determination of lappaconitine in mouse blood.

Development of a modified QuEChERS method based on magnetic multiwalled carbon nanotubes for the simultaneous determination of veterinary drugs, pesticides and mycotoxins in eggs by UPLC-MS/MS.

A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method was developed for the simultaneous determination of veterinary drugs, pesticides and mycotoxins in eggs by ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Several extraction conditions were tested and optimized, and the obtained extraction efficiency of all the targeted compounds (particularly macrolides) could fulfill the requirements. In the purification procedure, magnetic multiwalled carbon nanotubes (Fe3O4-MWCNTs) were used as adsorbents, and an external magnet was utilized to achieve a faster adsorbent separation, compared to the traditional centrifugation process. The recoveries of all analytes were in the range of 60.5%-114.6% at three fortified levels with relative standard deviations (RSDs) of less than 20%, and the LOQs ranged from 0.1 μg·kg-1 to 17.3 μg·kg-1. This method was successfully applied to the analysis of egg samples, demonstrating its applicability and suitability for the routine analysis of multiclass residues in egg samples.

Sensitive quantification of the somatostatin analog AP102 in plasma by ultra-high pressure liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study in rats.

AP102 is a di-iodinated octapeptide somatostatin agonist (SSA) designed to treat acromegaly and neuroendocrine tumors. A sensitive and selective method was validated for the quantification of AP102 in plasma following the European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. Sample preparation was performed using solid-phase extraction microplates. Chromatographic separation was achieved on an ultra-high pressure liquid chromatography (UHPLC) C18 column in 6.0minutes. The compounds were quantified using multiple reaction monitoring on a tandem quadrupole mass spectrometer with 13 C,15 N-labeled AP102 as internal standard. Calibration ranged from 50 to 10000pg/mL. The lower limit of quantification (LLOQ) was measured at 20pg/mL, and robust analytical performances were obtained with trueness at 99.2%-100.0%, intra-assay imprecision at 2.5%-4.4%, and inter-assay imprecision at 8.9%-9.7%. The accuracy profiles (total error) built on the 3 concentrations levels showed accuracy within the 70%-130% range. AP102 is remarkably stable since no proteolytic fragments were detected on plasma samples analyzed by Orbitrap-MS. Pharmacokinetic studies were conducted in rats, after single dose (1, 3, and 10 μg/kg, sc) and continuous subcutaneous administration (osmotic minipumps for 28days, 3.0 or 10.0 μg/kg/h). AP102 showed a rapid absorption by the subcutaneous route (Tmax : 15-30minutes) and a fast elimination (t1/2 : 33-86minutes). The PK profile of AP102 exhibited a mean clearance of 1.67L/h and a mean distribution volume at steady state of 7.16L/kg, about 10-fold higher than those observed with other SSA or non- and mono-iodinated AP102. LogD7.4 determination confirmed the lipophilic properties of AP102 that might influence its distribution in tissues.

Simultaneous determination of triamcinolone hexacetonide and triamcinolone acetonide in rabbit plasma using a highly sensitive and selective UPLC–MS/MS method

An ultra-high pressure liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was successfully developed and qualified for the simultaneous determination of triamcinolone hexacetonide (TAH) and triamcinolone acetonide (TAA, the active metabolite of TAH) in rabbit plasma. To prevent the hydrolysis of TAH to TAA ex vivo during sample collection and processing, we evaluated the effectiveness of several esterase inhibitors to stabilize TAH in plasma. Phenylmethanesulfonyl fluoride (PMSF) at 2.0 mM was chosen to stabilize TAH in rabbit plasma. The developed method is highly sensitive with a lower limit of quantitation of 10.0 pg/mL for both TAA and TAH using a 300 μL plasma aliquot. The method demonstrated good linearity, accuracy, precision, sensitivity, selectivity, recovery, matrix effects, dilution integrity, carryover, and stability. Linearity was obtained over the range of 10–2500 pg/mL. Both intra- and inter-run coefficients of variation were less than 9.1% and accuracies across the assay range were all within 100 ± 8.4%. The run time is under 5 minutes. The method was successfully implemented to support a rabbit pharmacokinetic study of TAH and TAA following a single intra-articular administration of TAH (Aristospan®).

Deposition of diazepam and its metabolites in hair following a single dose of diazepam.

Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10mg of diazepam. Hair was collected from all volunteers 1month after exposure, and also 2months post-exposure from men and 10months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.

Repeatability of gradient ultrahigh pressure liquid chromatography–tandem mass spectrometry methods in instrument-controlled thermal environments

The impact of viscous friction on eluent temperature and column efficiency in liquid chromatography is of renewed interest as the need for pressures exceeding 1000bar to use with columns packed with sub-2μm particles has grown. One way the development of axial and radial temperature gradients that arise due to viscous friction can be affected is by the thermal environment the column is placed in. In this study, a new column oven integrated into an ultrahigh pressure liquid chromatograph that enables both still-air and forced-air operating modes is investigated to find the magnitude of the effect of the axial thermal gradient that forms in 2.1×100mm columns packed with sub-2μm particles in these modes. Temperature increases of nearly 30K were observed when the generated power of the column exceeded 25W/m. The impact of the heating due to viscous friction on the repeatability of peak capacity, elution time, and peak area ratio to an internal standard for a gradient UHPLC-MS/MS method to analyze neurotransmitters was found to be limited. This result indicates that high speed UHPLC-MS/MS gradient methods under conditions of high viscous friction may be possible without the negative effects typically observed with isocratic separations under similar conditions.