Ultra-high Pressure Liquid Chromatography-tandem 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.

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.

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

SERUM CONC. OF ANTIHYPERTENSIVE DRUGS MAY REVEAL NON-ADHERENCE IN PATIENTS CONSIDERED TO HAVE TRUE TREATMENT RESISTANT HYPERTENSION ASSESSED BY DIRECTLY OBSERVED THERAPY

Objective: Detecting non-adherence is challenging. Both directly observed intake of antihypertensive medication (DOT) followed by 24 hour-ambulatory blood pressure monitoring and serum conc. measurements of antihypertensive drugs are objective methods considered reliable in revealing drug non-adherence. We aimed to investigate whether serum conc. offers additional information regarding drug adherence compared to DOT in patients with assumed true treatment resistant hypertension. Design and method: Patients referred for renal denervation (RDN) were thoroughly investigated and tested for adherence. 19 patients were defined as true treatment resistant when their daytime systolic BP remained high (> 135 mmHg) after DOT. Blood samples were collected at baseline, 6 months and 3 years after RDN and analyzed using ultra high pressure liquid chromatography-tandem mass spectrometry. Partial non-adherence was defined as at least one undetectable drug (but not all)and non-adherence as no detectable drugs in serum. Results: Ten out of 56 blood samples (17.9%) showed too low levels of one or more drug to be compatible with drug adherence. Six patients were redefined as either non-adherent or partially non-adherent based on their serum conc. results. Their systolic BPs are shown in the Figure. Three of these six patients went through a renal denervation procedure, while the remaining three were randomized to control. One of two patients considered to be RDN responders, became adherent to medication after six months which may have contributed to the fall in BP. At the 3 years visit none of the five antihypertensive agents prescribed were detected, and the patient́s BP was again elevated. Conclusions: Non-adherence was revealed in more patients when serum conc. measurements of antihypertensive drugs were added to directly observed therapy. Serum conc. measurement is objective and quantitative, and since some antihypertensive drugs require regular intake over a certain period of time to have maximal effect, they may offer important additional information.

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.

Rapid determination of 12 semi-volatile pesticide residues in vegetables by miniaturized thermal-assisted purge-and-trap coupled with ultra high pressure liquid chromatography-tandem mass spectrometry

A method for the analysis of 12 semi-volatile pesticide residues in vegetables was developed by miniaturized thermal-assisted purge-and-trap coupled with ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Under the optimized conditions, the sample preparation could be completed within 10 min. The linear relationships of the 12 semi-volatile pesticide residues were good within their corresponding concentration ranges. The correlation coefficients were in range of 0.9918-0.9997. The limits of detection and limits of quantification were in range of 0.3-1.3 μg/kg and 1.0-4.3 μg/kg, respectively. The recoveries ranged from 80.0% to 120.0%, with intra-day and inter-day RSDs of less than 10%. This proposed method is quick and simple, and is suitable for the simultaneous and rapid analysis of semi-volatile pesticide residues in vegetables.

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.

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.

Development and validation of rapid and simultaneous method for determination of 12 hair-growth compounds in adulterated products by UHPLC–MS/MS

Synthetic hair-growth compounds have been illegally used in diverse products to enhance the short-term efficacy of these products. In this study, a rapid and simultaneous method for the determination of hair-growth compounds in adulterated products based on ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) was developed and validated. The limit of detection (LOD) and limit of quantitation (LOQs) of the method were 0.08–43.6ng/mL and 0.27–145ng/mL for the solid-, liquid-, and cream-type samples, respectively. Good calibration linearity for all compounds was demonstrated with a correlation coefficient (r2) higher than 0.997. The intra- and inter-assay precisions were within 11%. The corresponding accuracies were 86–117% and 81–113%, respectively. The mean recoveries obtained for the solid-, liquid, and cream-type samples ranged from 87 to 114%, with a relative standard deviation (RSD) within 6%. The RSD of the stability evaluated at 4°C for 48h was less than 6%. The established method was used to screen 76 samples advertised as hair-growth treatments, from online and offline markets, over the course of two years. In 10% of the samples, four compounds, including triaminodil, minoxidil, finasteride, methyltestosterone, and testosterone–propionate were detected. The concentrations were in the range of 0.5–16.4mg/g. This technique provides a reliable platform for technical analysis for continuous monitoring of adulterated products to protect public health.

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.

High-throughput and rapid quantification of lipids by nanoflow UPLC-ESI-MS/MS: application to the hepatic lipids of rabbits with nonalcoholic fatty liver disease

A rapid and high-throughput quantification method (approximately 300 lipids within 20min) was established using nanoflow ultrahigh-pressure liquid chromatography-tandem mass spectrometry (nUPLC-ESI-MS/MS) with selective reaction monitoring (SRM) and applied to the quantitative profiling of the hepatic lipids of rabbits with different metabolic conditions that stimulate the development of nonalcoholic fatty liver disease (NAFLD). Among the metabolic conditions of rabbits in this study [inflammation (I), high-cholesterol diet (HC), and high-cholesterol diet combined with inflammation (HCI)], significant perturbation in hepatic lipidome (>3-fold and p < 0.01) was observed in the HC and HCI groups, while no single lipid showed a significant change in group I. In addition, this study revealed a dramatic increase (>2-fold) in relatively high-abundant monohexosylceramides (MHCs), sphingomyelins (SMs), and triacylglycerols (TGs) in both the HC and HCI groups, especially in MHCs as all 11 MHCs increased by larger than 3- to 12-fold. As the levels of the relatively high-abundant lipids in the above classes increased, the total lipidome level of each class increased significantly by approximately 2-fold to 5-fold. Other classes of lipids also generally increased, which was likely induced by the increase in mitogenic and nonapoptotic MHCs and SMs, as they promote cell proliferation. On the other hand, a slight decrease in the level of apoptotic ceramides (Cers) was observed, which agreed with the general increase in total lipid level. As distinct changes in hepatic lipidome were observed from HC groups, this suggests that HC or HCI is highly associated with NAFLD but not inflammation alone itself. Graphical Abstract Schematic of lipidomic analysis from hepatic tissue using nanoflow LC-ESI-MS/MS and nUPLC-ESI-MS/MS.

Validated UHPLC-MS/MS method for the simultaneous determination of pramipexole and ropinirole in plasma of patients with Parkinson's disease.

A simple and validated ultra high pressure liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of the dopaminergic agents pramipexole and ropinirole in plasma of patients with Parkinson's disease. Following liquid-liquid extraction with tert-butyl methyl ether from 250μL plasma, the separation of the analytes was achieved on a Gemini NX3 column using 10mM pH 6.0 ammonium formate and 10mM ammonium formate in methanol as binary gradient mobile phase at a flow rate of 0.3mL/min. The MS/MS ion transitions were 212.10→153.03 for pramipexole, 261.2→114.2 for ropinirole and 256.1→211 for the internal standard (lamotrigine). The lower limit of quantitation (LLOQ) for both analytes was 80pg/mL and the linearity was determined from 80 to 4000pg/mL for pramipexole and from 200 to 10000pg/mL for ropinirole. Mean recoveries were 94% for PRA and 73% for ROP. Both intra- and inter-assay precision and accuracy were ≤20% at LLOQ concentration and ≤15% at other concentrations. The proposed validated method was successfully applied to measure plasma concentrations of pramipexole and ropinirole in a series of patients with Parkinson's disease on chronic treatment. By grouping the two dopaminergic agents in the same assay, the method allows a large series of patient samples to be processed in a single analytical session.

An ultra-high pressure liquid chromatography-tandem mass spectrometry method for the quantification of teicoplanin in plasma of neonates

The development and validation of an ultra-high pressure liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was performed with the aim to be applied for the quantification of plasma teicoplanin concentrations in neonates. Pharmacokinetic data of teicoplanin in the neonatal population is very limited, therefore, a sensitive and reliable method for the determination of all isoforms of teicoplanin applied in a low volume of sample is of real importance. Teicoplanin main components were extracted by a simple acetonitrile precipitation step and analysed on a C18 chromatographic column by a triple quadrupole MS with electrospray ionization. The method provides quantitative data over a linear range of 25-6400ng/mL with LOD 8.5ng/mL and LOQ 25ng/mL for total teicoplanin. The method was applied in plasma samples from neonates to support pharmacokinetic data and proved to be a reliable and fast method for the quantification of teicoplanin concentration levels in plasma of infants during therapy in Intensive Care Unit.

Quantification of pravastatin acid, lactone and isomers in human plasma by UHPLC-MS/MS and its application to a pediatric pharmacokinetic study.

An ultra high pressure liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantitation of pravastatin and major metabolites, 3'α-hydroxy-pravastatin, pravalactone and 3'α-hydroxy-pravalactone, in human plasma has been developed and validated. Aliquots of (100μL) plasma in EDTA were diluted in pH 4.5 (0.1M buffer) to stabilize the analytes and subjected to hydrophilic lipophilic balance (HLB) solid phase extraction on 96 well μelution plates. Extracted samples were evaporated to dryness and reconstituted in pH 4.5 buffer. Chromatographic separation was performed on a Cortecs™ C18 column (2.1×100mm, 1.8μm), using gradient elution with a blend of acetonitrile and 10mM methylammonium acetate buffer (pH 4.5) at a flow rate of 0.4mL/min. Mass spectrometric detection was performed using multiple reaction monitoring (MRM) switching between positive/negative electrospay ionization (ESI). Pravastatin, 3'α-hydroxy-pravastatin, and internal standards [(2)H3]-pravastatin, and [(2)H3]-3'α-hydroxy-pravastatin were monitored in negative ESI mode at ion transitions m/z 423.2→321.1 and 426.2→321.1, respectively. Positive ESI mode was used for the detection of pravalactone, 3'α-hydroxy-pravalactone, and internal standards [(2)H3]-pravalactone, and [(2)H3]-3'α-hydroxy-pravalactone at ion transitions m/z 438.2→183.1 and 441.2→269.1 respectively. The method was linear for all analytes in the concentration range 0.5-200nM with intra- and inter-day precisions (as relative standard deviation) of ≤5.2% and accuracy (as relative error) of ≤8.0% at all quality control levels. The method was successfully applied to the investigation of pharmacokinetic properties of pravastatin and its metabolites in children after an oral dose of 20-40mg.

Stability-indicating UPLC-MS/UV Method for Simultaneous Determination of Sildenafil Citrate and Dapoxetine Hydrochloride from Bulk and Formulation

The present work was focused on development of rapid, specific and novel stability-indicating ultra-performance liquid chromatographic method for simultaneous determination of sildenafil citrate and dapoxetine hydrochloride in bulk and formulation. The drugs were subjected to hydrolysis (acidic, alkaline and neutral), photolytic and thermal stress, as per International Conference on Harmonisation guidelines Q1A (R2). The identification of major stressed degradation products was performed using qudrupole electrospray ionization mass spectroscopy. Separation of drugs from their degradation products was achieved on acquity ultra-performance liquid chromatographic I class equipped with photodiode array detector. Acquity BEH Shield RP18 column was used with the gradient elution and detection wavelength of 294 nm. The method was found to be linear in the concentration range of 2.5-100 µg/ml for both sildenafil and dapoxetine hydrochloride. Limit of detection and quantitation was found to be 0.15-0.45 μg/ml and 0.1-0.3 μg/ml for sildenafil and dapoxetine, respectively. Moreover, the fragmentation pattern of both drugs was studied using ultra-high pressure liquid chromatography-tandem mass spectrometry. Sildenafil was found to be unstable under acidic condition while dapoxetine was found to be unstable under alkaline conditions.

High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions.

Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry-based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography-tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example.

An improved method for retrospective quantification of sulfur mustard exposure by detection of its albumin adduct using ultra-high pressure liquid chromatography-tandem mass spectrometry.

Sulfur mustard (HD) adduct to human serum albumin (ALB) at Cys-34 residue has become an important and long-term retrospective biomarker of HD exposure. Here, a novel, sensitive, and convenient approach for retrospective quantification of HD concentration exposed to plasma was established by detection of the HD-ALB adduct using ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with a novel non-isotope internal standard (IS). The HD-ALB adduct was isolated from HD-exposed plasma with blue Sepharose. The adduct was digested with proteinase K to form sulfur-hydroxyethylthioethyl ([S-HETE])-Cys-Pro-Phe tripeptide biomarker. The tripeptide adduct could be directly analyzed by UHPLC-MS/MS without an additional solid phase extraction (SPE), which was considered as a critical procedure in previous methods. The easily available 2-chloroethyl ethylsulfide (2-CEES) as HD surrogate was first reported to be used as IS in place of traditional d8-HD for quantification of HD exposure. Furthermore, 2-CEES was also confirmed to be a good IS alternative for quantification of HD exposure by investigation of product ion spectra for their corresponding tripeptide adducts which exhibited identical MS/MS fragmentation behaviors. The method was found to be linear between 1.00 and 250 ng•mL(-1) HD exposure (R(2)>0.9989) with precision of <4.50% relative standard deviation (%RSD), accuracy range between 96.5% and 114%, and a calculated limit of detection (LOD) of 0.532 ng•mL(-1). The lowest reportable limit (LRL) is 1.00 ng•mL(-1), over seven times lower than that of the previous method. The entire method required only 0.1 mL of plasma sample and took under 7 h without special sample preparation equipment. It is proven to be a sensitive, simple, and rugged method, which is easily applied in international laboratories to improve the capabilities for the analysis of biomedical samples related to verification of the Chemical Weapon Convention (CWC).

Determination of urinary cortisol, cortisone and 6-sulfatoxymelatonin using dilute and shoot ultra-high pressure liquid chromatography-tandem mass spectrometry.

Human sleep is a natural part of every individual's life. Clear relationship between sleep and endocrine system has been already established. In particular, melatonin and cortisol are known to affect and regulate sleep/wake patterns. Here we report the development of an ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of 6-sulfatoxymelatonin (MT6s), cortisol and cortisone in urine. A separate method was developed for measurement of creatinine in urine. These levels were used to normalise the levels of analytes. First void morning urine samples were collected from 24 healthy volunteers. Samples were diluted 1:1 in water prior to injection onto reversed-phase C18 column and analysed using UHPLC-MS/MS method. Linear calibrations were obtained for all analytes with correlation coefficient in the range 0.998-0.999. The observed concentration was found to be in the range 92-105% for cortisol, 92-107% for cortisone and between 93 and 120% for MT6s of the reference levels. The total run time of 6 min with all peaks of interest eluting within 3 min was obtained. This demonstrates the feasibility of utilising the method for large multi-scale studies, where high throughput is required for studying the circadian rhythm of melatonin and cortisol secretion. These hormones play significant role in circadian rhythm and sleep/wake cycle; therefore it is important to monitor the levels of these endocrine markers in individuals suffering from sleep disorders. It is also beneficial with clinical applications to analyse melatonin and cortisol simultaneously in order to assess their interrelationships of these substances, such as their effect on diurnal rhythm and sleep.