Simultaneous Determination of Toxic Alkaloids in Blood and Urine by HPLC–ESI–MS/MS

A sensitive and selective method for simultaneous determination of 29 toxic alkaloids in human blood and 31 in urine using high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry was developed and validated. The samples were diluted with 0.1 mol L⁻¹ HCl, and the target alkaloids were purified by solid phase extraction. The separation of 31 alkaloids was carried out on a C18 column using a gradient mobile phase with 10 mmol L⁻¹ ammonium formate in water with 0.1% formic acid and methanol at the rate of 0.25 mL min⁻¹. The triple-quadrupole mass spectrometer equipped with an electrospray source in the positive mode was set up in the dynamic multiple reactions monitoring mode (dynamic MRM) to detect the ion transitions of 31 alkaloids. The calibration curves were linear over a range of 0.5–400, 1–400, or 4–400 μg L⁻¹ for target alkaloids in human blood and urine, and the correlation coefficients (r ²) was higher than 0.9943. The limit of determination and limit of quantification were 0.2–1 and 0.5–4 μg L⁻¹ for blood and urine, respectively. The only exceptions were sanguinarine and chelerythrine in human blood. All the target alkaloids were stable under the test condition. In addition, the solvent effect and reconstituted solution were investigated. The method was validated and proved to be accurate and precise over the studied concentrations and suitable for poisoning diagnosis and forensic toxicology.

     

Determination of cannabinoids in plasma using salting-out-assisted liquid–liquid extraction followed by LC–MS/MS analysis

The detection of the markers of Cannabis consumption in biological specimens is an important task for drug testing laboratories in varous contexts. A simple assay combining salting-out assisted liquid–liquid extraction sample preparation and LC–MS/MS analysis was applied to the measurement of Δ⁹-tetrahydrocannabinol, 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in 100 μl plasma specimens. The assay had linearity of 1–100 ng ml⁻¹ for THC-COOH and 0.5–50 ng ml⁻¹ for the other tested cannabinoids. Assay validation criteria were fulfilled. Extraction yields (88.7–97.3%) and internal-standard correct matrix effects (−9.6 to +5.4%) were acceptable. The assay was applied to 238 clinical specimens from trauma patients, with 19 samples presenting quantifiable concentrations of at least one of the target compounds. The developed assay is a simple and efficient strategy for simultaneous measurement of Δ⁹-tetrahydrocannabinol, THC-COOH, 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in plasma specimens.     

Determination of multiclass pesticides in food commodities by pressurized liquid extraction using GC–MS/MS and LC–MS/MS

Pressurized liquid extraction (PLE) was applied to the simultaneous extraction of a wide range of pesticides from food commodities. Extractions were performed by mixing 4 g of sample with 4 g of Hydromatrix and (after optimization) a mixture of ethyl acetate:acetone (3:1, v/v) as extraction solvent, a temperature of 100°C, a pressure of 1000 psi and a static extraction time of 5 min. After extraction, the more polar compounds were analyzed by liquid chromatography (LC), and the apolar and semipolar pesticides by gas chromatography (GC); in both cases LC and GC were coupled with mass spectrometry in tandem (MS/MS) mode. The overall method (including the PLE step) was validated in GC and LC according to the criteria of the SANCO Document of the European Commission. The average extraction recoveries (at two concentration levels) for most of the analytes were in the range 70–80%, with precision values usually lower than 15%. Limits of quantification (LOQ) were low enough to determine the pesticide residues at concentrations below or equal to the maximum residue levels (MRL) specified by legislation. In order to assess its applicability to the analysis of real samples, aliquots of 15 vegetable samples were processed using a conventional extraction method with dichloromethane, and the results obtained were compared with the proposed PLE method; differences lower than 0.01 mg kg⁻¹ were found.     

Determination of arsenic species and arsenosugars in marine samples by HPLC–ICP–MS

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP–MS detection. Separation of eight arsenic species—As^III, MMA, DMA, As^V, AB, TMAO, AC and TeMAs⁺—was achieved on a C₁₈ column with isocratic elution (pH 3.0), under which conditions As^III and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC–ICP–MS detection limits for the eight arsenic species were in the range 0.03–0.23 μg L⁻¹ based on 3σ for the blank response (n=5). The precision was calculated to be 2.4–8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18–9.59 μg g⁻¹. This paper was presented at the European Winter Conference 2005     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of steroid hormones in blood by GC–MS/MS

This paper presents the development, optimization and validation of a methodology to determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) expressed in the steroidogenesis in biological fluids. The analytical method allows for the determination of steroid hormones in blood plasma and serum down to 0.08–0.16 ng/mL for estrogens, 0.20–0.36 ng/mL for androgens and 0.36–0.43 ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-phase clean-up for fractionation and elimination of interfering lipids (fatty acids, phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the method was 50–112% in the range 0.10 to 2.00 ng/mL.     

Determination of chlormequat in pig serum and sow milk by LC–MS/MS

Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC–MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol–water–acetic acid, centrifuged, filtrated and determined by LC–MS/MS. The mean recoveries were in the range 80–110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g−4.0 ng/g.     

Identification and Determination of Related Substances in Diosmin Bulk Drug and Pharmaceutical Formulations by HPLC and HPLC–MS

Six related substances were detected in diosmin bulk drug substances and products by a newly developed gradient reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection. The chromatographic system consisted of an Intersil Wondasil TM ODS (C 18) column (250 × 4.6 mm; 5 μm). The mobile phase consisted of water/acetic acid 66:6 v/v (solvent A) and methanol (solvent B) using a gradient program at a flow rate of 0.8 mL min^?1 with 345 nm detection and an injection volume of 20 μL. In addition, the linearity, quantitation limit (QL), accuracy, selectivity, robustness and precision were determined. Good linearity was obtained over the concentration range 0.5–200 μg mL^?1 with the coefficient of determination (r ²) of 0.999. The QL was 0.125 μg mL^?1 (relative standard deviation <2.0 %). The major impurities have been resolved and identified using two analytical systems, HPLC and HPLC/electrospray ionization-mass spectrometry operated in a negative ion mode. One of these impurities marked as 7-hexopyranosidal diosmetin was unknown and has not been reported previously. Based on mass spectrometry data the structure of the new impurity was proposed as 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-4H-chromen-7-yl hexopyranoside. The newly developed RP–LC method for quantitative determination of diosmin-related substances was found to be precise, accurate, robust and specific. It has been successfully employed for the quality evaluation of different sources of raw material and generic formulations of diosmin.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Simultaneous Determination of Fluoroquinolones, Tetracyclines and Sulfonamides in Chicken Muscle by UPLC–MS–MS

A rapid and sensitive analytical method for the simultaneous determination of four fluoroquinolones, four tetracyclines and six sulfonamides in chicken muscle using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS–MS) has been developed and validated. Samples were extracted with McIlvaine buffer-acetonitrile, defatted with n-hexane, and analyzed by UPLC–MS–MS. Solvent delay technique was applied in the analysis to remove the non-volatile phosphate and carry out farther on-line SPE clean-up. Satisfactory recoveries (55–110%) of all the veterinary drugs were demonstrated in 1, 10 and 20 μg kg^?1 spiked levels with the overall RSD for intra- and inter-day of 14 analytes less than 18%. The LOD and LOQ were 0.3 and 1.0 μg kg^?1, respectively. Quantitative results of 103 real samples indicated that the present method was suitable for the quantitative analysis of real samples.     

Simultaneous Determination of 1-Methyltryptophan and Indoleamine 2,3-Dioxygenase Biomakers of Tryptophan and Kynurenine in Mice Tumors by HPLC–MS/MS

Indoleamine 2,3-dioxygenase (IDO), an immune checkpoint protein, can cause the depletion of tryptophan (Trp) and accumulation of its metabolite of kynurenine (Kyn) in cancer cells, and generates the immunosuppressive microenvironment that supports tumor cell growth. A novel immunoregulatory prodrug micelle based on polyethylene glycol-derivatized an IDO-selective inhibitor of 1-methyltryptophan (1-MT), PEG-Fmoc-1-MT, was developed for inhibiting the IDO activity of the conversion of Trp to Kyn in tumor microenvironments. To investigate the 1-MT distribution and Trp/Kyn ratios in mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment, a HPLC–MS/MS method for simultaneous determination of 1-MT and IDO biomakers of Trp and Kyn in mouse tumors was developed and validated. Triple-quadrupole mass spectrometry with positive electrospray ionization as source ionization in multiple reaction monitoring at m/z 219.0?→?160.1, 205.0?→?118.2, 209.0?→?146.1 and 249.3?→?148.3 was used for determination of 1-MT, Trp, Kyn and matrine (internal standard). The method demonstrated good linearity at the concentrations ranging from 10 to 10,000 ng/mL and lower limits of quantitation of 1 ng/mL for 1-MT, Trp and Kyn, respectively. The validated method was successfully applied to 1-MT tumor biodistribution and Trp/Kyn ratio studies in 4T1 tumor bearing mice i.v. with PEG-Fmoc-1-MT prodrug micelles. The mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment exhibited higher 1-MT accumulation and lower Trp/Kyn ratio, in comparison with those of mice with 1-MT solution treatment. The developed PEG-Fmoc-1-MT prodrug micelles could be a promising IDO immunoregulatory prodrug micelles for cancer immunotherapy.

     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Quantification of multi-class antibiotics by UHPLC–MS/MS analysis combined with salt-assisted acetonitrile extraction: comparative evaluation of dairy and poultry manure

ABSTRACT

A comprehensive multiresidue method for the analysis of 33 antibiotics from 7 prevalent classes was comparably investigated for both dairy and poultry manure samples, which can be important pollution sources for the release of antibiotics into the environment. Following salting-out-assisted extraction with acetonitrile, the antibiotics were quantified with ultrahigh-performance liquid chromatography tandem mass spectrometry without a clean-up step. By changing the composition of the mobile phase for chromatography, a pronounced signal enhancement was achieved not only for tetracyclines (TCs) but also for other groups of antibiotics in the manure samples. Although the physicochemical properties of selected antibiotics were quite different, the apparent recovery values from dairy and poultry manure samples by using an extraction solvent comprising McIlvaine buffer and ethylenediaminetetraacetic solution at pH 3 were 86–121% and 89–113%, respectively. Apparent recovery of the antibiotics was not remarkably affected by the extraction solvent over a wide range of pH values, with the exception of the recovery of TC antibiotics from poultry manure, which was in the 53–55% range at pH 8. Furthermore, the poor performance of the analytical method for a few of the antibiotics in poultry manure was correlated with high metal and organic contents of the complicated matrix. The high suppression effects of co-eluted matrix components were compensated by constructing matrix-matched calibration curves and by using internal standards. Simultaneous quantification of seven different antibiotic classes with low limit of detection values varying from 0.38 to 31 μg kg^?1 for dairy manure and from 0.32 to 5.85 μg kg^?1 for poultry manure facilitated their monitoring. The application of the developed analytical method to dairy, broiler and layer-hen manure samples from confined animal feeding operations showed that a wide variety of antibiotics at high concentrations were found in broiler manure.     

Determination of nitrophenolate sodium in aquatic products by HPLC–MS/MS with atmospheric pressure chemical ionization

The paper describes an efficient method for the determination of nitrophenolate sodium in aquatic products by HPLC combined with atmospheric pressure chemical ionization with tandem mass spectrometry (LC–APCI-MS/MS). Analytes were extracted from aquatic products by acetonitrile, the extracts were degreased by alumina and concentrated, the concentrated solution was further purified by Oasis HLB cartridge. Finally, the analytes were separated and detected by LC–APCI-MS/MS in negative ion mode. Excellent linearity with correlation coefficients of more than 0.995 was observed in the concentration range of 2–200 μg/L for p-nitrophenol sodium and 2-methoxy-5-nitrophenolate sodium, and 5–200 μg/L for ο-nitrophenol sodium. Recovery rates of nitrophenolate sodium between 86.1–94.3% were achieved. Limit of quantitation of p-nitrophenol sodium and 2-methoxy-5-nitrophenolate sodium was 2 μg/kg and ο-nitrophenol sodium was 5 μg/kg, with relative standard deviations <10%. This method was employed in the practical analysis of spiked and naturally contaminated aquatic products.     

Determination of oleandrin and adynerin in rat plasma by UPLC–MS/MS and their pharmacokinetic study

Oleandrin and adynerin are the main toxic components of oleander, an evergreen shrub or a small tree of the oleander family, which belongs to the class of cardiac glycosides exhibiting delayed action. The pharmacokinetic differences of oleandrin and adynerin in rats were studied by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) under two different administration modes: oral (5 mg/kg) and sublingual intravenous injection (1 mg/kg). The chromatographic column was UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm), and the column temperature was set at 40 °C. The mobile phase was acetonitrile–water (containing 0.1 % formic acid), with gradient elution, the flow rate was 0.4 mL/min, and the elution time was 4 min. Electrospray (ESI) positive ion mode detection with multiple reaction monitoring mode (MRM) was used for quantitative analysis: oleandrin m/z 577 → 145, adynerin m/z 534 → 113, and internal standard m/z 237 → 135. The established UPLC–MS/MS method was successfully applied to the pharmacokinetics in rats after administering oleandrin and adynerin. The bioavailability of oleandrin and adynerin was found to be low, 7.0 % and 93.1 %; respectively.