Ultra-performance Liquid Chromatography Separation Research Articles

Chiral pharmacokinetics of ibuprofen enantiomers in Chinese preterm neonates with patent ductus arteriosus using a validated UHPLC-MS/MS method

Persistent patent ductus arteriosus (PDA) is generally observed in preterm neonates. Oral ibuprofen is the standard treatment for closing PDA in China. To investigate the chiral pharmacokinetics of ibuprofen enantiomers in Chinese premature infants with PDA, a simple, fast, and sensitive analytical enantioselective technology was developed with ultra-performance liquid chromatography (UPLC) – tandem mass spectrometry (MS/MS). Chromatographic separation of (R)-ibuprofen and (S)-ibuprofen was accomplished on a Lux® 3 µm Cellulose-3 (150 mm × 2.0 mm, 3 μm) at a flow rate of 0.2 mL/min within 6 min. UPLC separation was achieved by isocratic elution with a mobile phase consisting of formic acid:water (75:1000000, v/v) and acetonitrile:methanol (1:1, v/v). Only 50 µL of plasma samples were pre-treated with acetonitrile precipitation. Ibuprofen-d3 was used as an internal standard. The standard curves of both enantiomers were linear over a concentration range of 0.0500 μg/mL to 50.00 μg/mL. The method has been validated for selectivity, carryover effect, lower limit of quantification, precision, accuracy, matrix effect, extraction recovery, dilution integrity, and stability based on the existing guidelines of the National Medical Products Administration, the United States Food and Drug Administration, and the European Medicines Agency. This method has been successfully applied to investigate the pharmacokinetics of ibuprofen enantiomers in 9 preterm infants with PDA. Our results showed that a high chiral inversion ratio of (R)- to (S)-ibuprofen exists in Chinese preterm neonates. Further studies should be conducted to monitor drug concentration following oral administration of ibuprofen and to consider the effect of individual variations and ethnic differences in metabolizing enantiomers of ibuprofen in premature neonates with PDA.

Application of solid-phase extraction: High-resolution mass spectrometry analysis strategy in the characterization and quantification of amide herbicides in aquatic products.

In this study, we developed a new method combining thin-layer ultrasonic extraction, efficient SPE purification, ultra-performance liquid chromatography separation, and high-resolution mass spectrometry characterization for seven amide herbicides in fishery products. In sample preparation, to rapidly increase the contact area between the fish meat and the extractant, the fish meat was smeared on a glass slide. This process resulted in quickly reaching the extraction equilibrium and relatively high extraction efficiency. In data analysis, a strategy for characterization and qualitative analysis was constructed by analyzing the fragmentation of amide herbicides using product ion scans. Isomeric pretilachlor and butachlor were separated chromatographically, while the coeluting isomers, alachlor and acetochlor, could be separated by differences in the fragmentation of their selected precursor ions. This method overcame the challenge of poor dispersion in the extractant caused by the high viscosity of fish meat, and the challenge of separation and characterization for isomers. Compared with other methods, the extraction efficiency was improved and the amide herbicides in aquatic products was characterized and quantified rapidly and accurately. Moreover, the qualitative information was much greater and provided an additional strategy for analytes identification. This rapid and accurate method will benefit workers involved in monitoring fishery.

Infrared laser ablation sampling coupled with data independent high resolution UPLC-IM-MS/MS for tissue analysis

Infrared laser ablation microsampling was used with data-dependent acquisition (DDA) and ion mobility-enhanced data-independent acquisition (HDMSE) for mass spectrometry based bottom-up proteomics analysis of rat brain tissue. Results from HDMSE and DDA analyses of the 12 laser ablation sampled tissue sections showed that HDMSE consistently identified approximately seven times more peptides and four times more proteins than DDA. To evaluate the impact of ultra-performance liquid chromatography (UPLC) peak congestion on HDMSE and DDA analysis, whole tissue digests from rat brain were analyzed at six different UPLC separation times. Analogous to results from laser ablated samples, HDMSE analyses of whole tissue digests yielded about four times more proteins identified than DDA for all six UPLC separation times.

Rapid quantification and isotopic analysis of dissolved sulfur species.

Dissolved sulfur species are of significant interest, both as important substrates for microbial activities and as key intermediaries in biogeochemical cycles. Species of intermediate oxidation state such as sulfite, thiosulfate, and thiols are of particular interest but are notoriously difficult to analyze, because of low concentrations and rapid oxidation during storage and analysis. Dissolved sulfur species are reacted with monobromobimane which yields a fluorescent bimane derivative that is stable to oxidation. Separation by Ultra-Performance Liquid Chromatography (UPLC) on a C18 column yields baseline resolution of analytes in under 5min. Fluorescence detection (380nm excitation, 480nm emission) provides highly selective and sensitive quantitation, and Time-of-Flight Mass Spectrometry (TOF-MS) is used to quantify isotopic abundance, providing the ability to detect stable isotope tracers (either 33 S or 34 S). Sulfite, thiosulfate, methanethiol, and bisulfide were quantified with on-column detection limits of picomoles (μM concentrations). Other sulfur species with unshared electrons are also amenable to analysis. TOF-MS detection of 34 S enrichment was accurate and precise to within 0.6% (relative) when sample and standard had similar isotope ratios, and was able to detect enrichments as small as 0.01 atom%. Accuracy was validated by comparison to isotope-ratio mass spectrometry. Four example applications are provided to demonstrate the utility of this method. Derivatization of aqueous sulfur species with bromobimane is easily accomplished in the field, and protects analytes from oxidation during storage. UPLC separation with fluorescence detection provides low-μM detection limits. Using high-resolution TOF-MS, accurate detection of as little as 0.01% 34 S label incorporation into multiple species is feasible. This provides a useful new analytical window into microbial sulfur cycling. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous Ultra Performance Liquid Chromatography Determination and Antioxidant Activity of Linarin, Luteolin, Chlorogenic Acid and Apigenin in Different Parts of Compositae Species.

Linarin (LA), luteolin (LE), chlorogenic acid (CA) and apigenin (AP) are four major flavonoids with various promising bioactivities found in Compositae (COP) species. A reliable, reproducible and accurate method for the simultaneous and quantitative determination of these four major flavonoids by Ultra Performance Liquid Chromatography (UPLC) analysis was developed. This method should be appropriate for the quality assurance of COP. The UPLC separation was carried out using an octadecylsilane (ODS) Hypersil (2.1 mm × 250 mm, 1.9 μm) and a mobile phase composed of acetonitrile and 0.1% formic acid in water at a flow rate 0.44 mL/min and ultraviolet (UV) detection 254 nm. Gradient elution was employed. The method was precise, with relative standard deviation below 3.0% and showed excellent linearity (R2 > 0.999). The recoveries for the four flavonoids in COP were between 95.49%–106.23%. The average contents of LA, LE, CA and AP in different parts (flower, leave and stem) of COP were between 0.64–1.47 g/100 g, 0.66–0.89 g/100 g, 0.32–0.52 g/100 g and 0.16–0.18 g/100 g, respectively. The method was accurate and reproducible and it can provide a quantitative basis for quality control of COP.

Extraction optimization of gallic acid, (+)-catechin, procyanidin-B2, (–)-epicatechin, (–)-epigallocatechin gallate, and (–)-epicatechin gallate: their simultaneous identification and quantification in Saraca asoca

The objective of the present investigation was to optimize extraction conditions for maximum recovery of bioactive phenolics from different parts of Saraca asoca. Extraction recovery was optimized using a mixture of methanol and water in different proportions. For identification and quantification of six analytes, a rapid reversed phase ultra-performance liquid chromatography (UPLC) photo diode array detection method was developed. UPLC separation was achieved in a gradient elution mode on a C18 column with acetonitrile and aqueous phosphoric acid (0.1%, pH = 2.5). Extraction solvent for maximum recovery of analytes varied depending on the nature of matrices. The developed UPLC method was validated in accordance with International Council for Harmonisation (ICH) guidelines. Wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase composition implied that the method could be suitable for routine analysis of all six analytes with high precision and accuracy. The uniqueness of this study is the determination of the distribution of these compounds in the various parts of S. asoca.

Quantitative Analysis of Melissa officinalis L. Samples by Chromatographic Multivariate Calibration Methods

In this study, improved chromatographic multivariate calibration methods were developed for the quantitative analysis of caffeic acid (CAA) and rosmarinic acid (RA) in 12 different plant samples of Melissa officinalis L. (lemon balm), a perennial herb which belongs to the Lamiaceae family and is a well-known medicinal plant. UPLC (ultra performance liquid chromatography) separation was performed on a Waters UPLC BEH phenyl column (100 mm × 2.1 mm i.d., 1.7 μm) using a mobile phase consisting of acetonitrile, 0.1 M formic acid buffer (pH 3.80), and acetic acid buffer (pH 4.74) (17:76:7, v/v) in the presence of 1.0 mL triethylamine in 1000 mL mobile phase. Multiwavelength chromatograms were obtained by photodiode array (PDA) detection in the wavelength range 275–345 nm at intervals of Δλ = 10 nm. Partial least squares (PLS) and principal component regression (PCR) analyses were utilized by applying PCR and PLS algorithms to the concentration set and to the multiwavelength peak areas for both CAA and RA. The proposed UPLC-PCR and UPLC-PLS methods permitted the accurate quantification of CAA and RA in 12 samples of M. officinalis obtained from different herbalists in several Turkish cities.

Integration of electrochemistry with ultra-performance liquid chromatography/mass spectrometry.

This study presents the development of ultra-performance liquid chromatography (UPLC) mass spectrometry (MS) combined with electrochemistry (EC) for the first time and its application for the structural analysis of proteins/peptides that contain disulfide bonds. In our approach, a protein/peptide mixture sample undergoes a fast UPLC separation and subsequent electrochemical reduction in an electrochemical flow cell followed by online MS and tandem mass spectrometry (MS/MS) analyses. The electrochemical cell is coupled to the mass spectrometer using our recently developed desorption electrospray ionization (DESI) interface. Using this UPLC/EC/DESI-MS method, peptides that contain disulfide bonds can be differentiated from those without disulfide bonds, as the former are electroactive and reducible. MS/MS analysis of the disulfide-reduced peptide ions provides increased information on the sequence and disulfide-linkage pattern. In a reactive DESI- MS detection experiment in which a supercharging reagent was used to dope the DESI spray solvent, increased charging was obtained for the UPLC-separated proteins. Strikingly, upon online electrolytic reduction, supercharged proteins (e.g., α-lactalbumin) showed even higher charging, which will be useful in top- down protein structure MS analysis as increased charges are known to promote protein ion dissociation. Also, the separation speed and sensitivity are enhanced by approximately 1(~)2 orders of magnitude by using UPLC for the liquid chromatography (LC)/EC/MS platform, in comparison to the previously used high- performance liquid chromatography (HPLC). This UPLC/EC/DESI-MS method combines the power of fast UPLC separation, fast electrochemical conversion, and online MS structural analysis for a potentially valuable tool for proteomics research and bioanalysis.

Ultra-Performance Liquid Chromatography for the Simultaneous Quantification of Rutin and Chlorogenic Acid in Leaves of Ribes L. Species by Conventional and Chemometric Calibration Approaches.

A new ultra-performance liquid chromatography (UPLC) using conventional and chemometric calibrations was improved for the simultaneous estimation of chlorogenic acid (CA) and rutin (RUT) in leaves of Ribes L. species (R. rubrum, R. biebersteinii, R. nigrum, R. uva-crispa, R. alpinum, R. orientale, R. multiflorum and R. anatolica). The UPLC separation for CA and RUT in samples were performed on a Waters UPLC BEH phenyl column (100 mm × 1.0 mm i.d., 1.7 μm) and mobile phase consisting of acetonitrile and 0.1 M formic acid buffer, pH = 3.77 (15:85, v/v) containing 1.0 mL triethylamine in 1,000 mL mobile phase. Multi-wavelength UPLC chromatograms of CA and RUT in calibration and plant samples were obtained by the photodiode array (PDA) detection at the wavelength set from 290 to 360 nm with the interval of Δλ = 10 nm. Conventional UPLC-single and chemometric calibrations were subjected to the analysis of the related compounds. By using UPLC data, conventional and chemometric calibrations in the linear concentration range between 2.5 and 40.0 μg/mL for all compounds were applied for the simultaneous quantitative analysis of CA and RUT in plant samples of seven different Ribes species.

Ultra-performance liquid chromatography coupled with graphene/polyaniline nanocomposite modified electrode for the determination of sulfonamide residues.

An ultra-performance liquid chromatography (UPLC) coupled with graphene/polyaniline (G/PANI)-modified screen-printed carbon electrode was developed for separation and sensitive determination of eight sulfonamides (SAs) in shrimp. Electrospraying was selected for electrode modification because it can generate the well dispersion of G/PANI nanocomposites on the electrode surface. Prior to electrochemical detection, eight SAs were completely separated within 7 min by using reversed phase UPLC (C4) with mobile phase containing 70:25:5 (v/v/v) of potassium hydrogen phosphate (pH 3):acetonitrile:ethanol. For amperometric detection, the detection potential acquired from hydrodynamic voltammetry was found to be +1.4V. Under optimal conditions, a wide linearity and low limit of detection were obtained for eight SAs in the range of 0.01-10 µg mL(-1) and 1.162-6.127 ng mL(-1), respectively. Compared to boron-doped diamond (BDD) electrode, a G/PANI-modified screen-printed carbon electrode offered higher sensitivity with lower operating cost. To determine SAs in shrimp samples, solid-phase extraction was used to clean up and preconcentrate the samples prior to UPLC separation. To validate this developed method, a highly quantitative agreement was accomplished with UPLC-UV system. Thus, this proposed system might be an alternative approach for rapid, inexpensive, and sensitive determination of SAs in shrimps.

Analysis of maltooligosaccharides in honey samples by ultra-performance liquid chromatography coupled with evaporative light scattering detection

We report the use of ultra performance liquid chromatography (UPLC) separation together with evaporative light scattering detection (ELSD) to effectively distinguish honey adulterated with maltooligosaccharides. UPLC separation utilizing a mobile phase with pure water and acetonitrile containing 0.2% triethylamine, resulted in enhanced separation and a running time of less than 6min. Also, only a simple liquid–liquid extraction was required for the sample pretreatment. This UPLC–ELSD method provides limits of quantifications (LOQs) from 0.30 to 0.60mg/g for the six maltooligosaccharides (maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose) ranging from 15 to 2000μg/mL with excellent linearity(R2≥0.9914). This optimized method also offers a satisfactory intra-day precision of 2.4%–9.4%, inter-day precision of 5.5%–12.1% and accuracy of −7.7%–+11.4% at each level. We successfully applied this system for detecting maltooligosaccharide contamination in 290 honey samples collected from apiaries, supermarkets and traditional agri-product markets. Concentrations of the six maltooligosaccharides varied from undetectable to 14.1mg/mL. The lowest quantifiable concentration that could be detected by this established method was 0.132mg/mL. This study therefore demonstrates the reliability and potential use of the UPLC–ELSD technique in scrutinizing the authenticity of honey in the food processing industry.

Simultaneous determination of statins in dietary supplements by ultra-performance liquid chromatography

A method for the determination of 12 statins [atorvastatin (ATOR), cerivastatin (CERI), fluvastatin (FLU), lovastatin (LO), lovastatin acid (LOA), mevastatin (ME), mevastatin acid (MEA), pitavastatin (PITA), pravastatin (PRA), rosuvastatin (ROSU), simvastatin (SIM), and simvastatin acid (SIMA)] in dietary supplements by ultra-performance liquid chromatography (UPLC) has been developed. Statins were ultrasonically extracted with 50% (v/v) methanol. Clean-up was performed using an Oasis MAX mini-cartridge column with methanol and methanol containing 0.2% (v/v) phosphoric acid as an eluting solvent. UPLC separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm i.d. × 150 mm, 1.7 μm) with 0.2% (v/v) phosphoric acid aqueous solution-acetonitrile gradient. The method was validated for dietary supplements spiked with the 12 statins at the quantitation limits and 10 times the quantitation limits, and the recoveries of statins were between 89.2% and 100.9%. Relative standard deviation values of repeatability and intermediate precision were not more than 7%. The analytical method was applied to 24 commercial dietary supplements. LO and LOA were found at maximum concentrations of 4.85 mg/packet and 1.28 mg/capsule, respectively. Other statins were not detected. When a dietary supplement was consumed according to the directions on the package, the daily intake of LO was 6.74 mg. This could be dangerous to consumers because it exceeds one half of the lowest recommended daily dose of LO (10 mg).

Rapid Characterization of Wheat Low Molecular Weight Glutenin Subunits by Ultraperformance Liquid Chromatography (UPLC)

Low molecular weight glutenin subunits (LMW-GS), as important seed storage proteins, together with HMW-GS significantly define unique dough viscoelastic properties. In this study, a rapid ultraperformance liquid chromatography (UPLC) method for the separation and characterization of LMW-GS in wheat was optimized and established. The fast, reproducible, and high-resolution UPLC separation of LMW-GS could be obtained by gradually increasing eluting gradient from 21 to 47% in 30 min at flow rate of 0.55 mL/min and 60 °C for separation temperature. By this method, analysis of one sample could be completed in <20 min, significantly less time than the traditional reversed-phase high-performance liquid chromatography (RP-HPLC) method. Under the optimized conditions, the genetic features of LMW-GS and genotype × environmental interaction were successfully analyzed, leading to a fast identification of 17 main LMW-GS alleles that were related to different quality properties in wheat. The results demonstrated that UPLC could be a powerful and alternative tool for genetic and proteomic studies of wheat grain proteins and fast identification or screening of desirable LMW-GS alleles in wheat quality improvement.

Technical note: Development and validation of a method using ultra performance liquid chromatography coupled with tandem mass spectrometry for determination of vitamin B12 concentrations in milk and dairy products

A method using ultra performance liquid chromatography coupled with tandem mass spectrometry was developed to measure cobalamins in naturally enriched raw milk and to evaluate their fate during thermal treatments and along the process of cheese making. After addition of methotrexate as internal standard, samples were submitted to heat treatment in the presence of cyanide, which converts all the less-stable cobalamins into cyanocobalamin; then, purification was performed by a solid-phase extraction step. Reverse-phase ultra performance liquid chromatography separation coupled with tandem mass spectrometry provided a fast and reliable determination. Mass spectrometric analysis was carried out in multiple reaction monitoring mode. The monitored transitions were m/z 678.36 → 147.10 and 678.36 → 359.30 for vitamin B12 and m/z 455.22 → 175.13 and 455.22 → 308.22 for methotrexate (internal standard). The limit of quantification was 2ng/g. The method showed good linearity from 2 to 20ng/g (R2 ≥0.98) and intra- and interday precisions were always less than 19%.

Determination of 17 amino acids in fish eggs by ultra performance liquid chromatography coupled with precolumn derivatization

A rapid quantitative method of ultra performance liquid chromatography (UPLC) has been developed for the analysis of 17 amino acids in fish eggs from Acipenser schrenckii, Huso dauricus and Acipenser ruthenus. The analytes were hydrolyzed with 6 0 mol/L HCI. The extraction solution was concentrated under low pressure and neutralized with NaOH solution before derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) as derivatization agent in borate buffer solution (pH 8.8). Gradient UPLC separation was performed on a C, column (BEH C18, 100 mm x2.1 mm, 1.7 micro m) with 30 mmol/L ammonium acetate (pH 3. 5 adjusted with formic acid) and acetonitrile (containing 0. 15% (v/v) formic acid and 30 mmol/L ammonium acetate) as the mobile phases at a flow rate of 0. 7 mL/min. The detection was carried out with a photo-diode array (PDA) detector and the wavelength was set at 260 nm. The linear ranges were from 5.0 to 1000 micro mol/L with the correlation coefficients (r2) > or = 0. 995 0. The method was validated by the analysis of seven replicates. The 17 amino aci standards were spiked in fish eggs at three levels of 100, 500 and 750 micro mol/L, and the averag recoveries were 75.4% - 107.3% with the relative standard deviations of 2. 19% - 12.3%. The limits of detection (LOD) for the analytes were from 0.94 micro mol/L to 4.04 micro mol/L. The method was successfully applied to the analysis of the 17 amino acids in fish eggs from Acipenser schrenckii, Huso dauricus and Acipenser ruthenus. The method is simple, rapid, precise and reliable.

Determination of thyroid hormones and their metabolites in tissue using SPE UPLC‐tandem MS

A solid-phase liquid chromatography tandem mass spectrometry (SPE LC-MS/MS) method was developed to determine thyroid hormones and their metabolites in tissue samples. The separation was achieved using reversed-phase ultra-performance liquid chromatography (UPLC); the mass spectrometric detection was achieved by positive electrospray ionization and multiple reaction monitoring. Prior to the UPLC separation a sample cleanup with a cation exchange was performed. ¹³C₆ labeled internal standards were used for the thyroid hormones and their metabolites. The method was linear over a range from 0.23 to 90 nmol/L for thyroxine and from 0.23 to 9 nmol/L for the metabolites. The lower limit of quantification ranged from 0.98 to 1.73 pg on column. Intra- and total assay variation were <10 and <15%, respectively. This method enables us to link thyroid hormone tissue concentrations to local iodothyronine deiodinase expressions, which will enhance our understanding of the regulation of thyroid hormone metabolism on the tissue level.

A Method for Comprehensive Analysis of Urinary Acylglycines by Using Ultra-Performance Liquid Chromatography Quadrupole Linear Ion Trap Mass Spectrometry

Acylglycines are an important class of metabolites that have been used in the diagnosis of several inborn errors of metabolism (IEM). However, current analytical methods detect only a few acylglycines. There is a need to profile these metabolites in a comprehensive manner for studying their functions and improving their diagnostic values for different IEM and potentially other diseases. We describe a sensitive method that combines the chromatographic resolving power of ultra-performance liquid chromatography (UPLC) to separate closely related metabolites including isomers with tandem mass spectrometry (MS/MS). Acylglycines were extracted from urine using an anion exchange solid-phase extraction (SPE) cartridge. After UPLC separation, the acylglycines were detected on a hybrid triple quadrupole linear ion trap mass spectrometer. A set of standards were used for the development of an optimal MS acquisition method. Several acquisition modes using information derived from collision-induced dissociation breakdown curves were used to detect acylglycines. Using this method, 18 acylglycines were detected in the urine of healthy individuals and confirmed using standards, while 47 additional acylglycines were detected and tentatively identified, based on their retention and fragmentation pattern. Among the 65 acylglycines detected, only 18 of them have been previously reported in biofluids of healthy individuals. These results will be deposited in a public human metabolome database. This example illustrates that by developing a method tailored to the analysis of a class of metabolites sharing similar structural moieties, we can potentially identify many more new metabolites, thereby expanding the overall metabolome coverage.

Validation of a Method for the Determination of Chloramphenicol in Poultry and Swine Liver by Ultra-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry

A sensitive and reliable method has been developed and validated for the determination of chloramphenicol in poultry and swine liver using SPE and ultra-performance liquid chromatography (UPLC)/MS/MS. The liver samples were extracted with ethyl acetate, defatted with n-hexane, and further cleaned up using SPE cartridges with polymeric sorbent. An Acquity BEH C18 column was used for gradient UPLC separation, with water and acetonitrile as the mobile phase. The multiple reaction monitoring mode was used for two precursor-product ion transitions for chloramphenicol and one for the internal standard. The method was validated at 0.1, 0.3, and 1.0 microg/kg. Mean recoveries from fortified samples ranged from 95.5 to 106.7% with an RSD of 12.2%. The method LOD was < 0.02 microg/kg.

Rapid, sensitive and simultaneous determination of fluorescence-labeled designated substances controlled by the Pharmaceutical Affairs Law in Japan by ultra-performance liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry

A simultaneous determination method based on ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) was developed for 16 "designated substances" (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan. These substances were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole at 60 degrees C for 2 h in 0.1 M borax (pH 9.3). The resulting fluorophores were well separated by reversed-phase chromatography using an Acquity UPLC BEH C(18) column (1.7 microm, 100 mm x 2.1 mm i.d.) by isocratic elution with a mixture of water and acetonitrile-methanol (20:80) containing 0.1% formic acid. The separated derivatives were sensitively detected by both FL and TOF-MS. However, the determination of several designated substances by FL detection showed interference from endogenous substances in biological samples. Therefore, the determination in real samples was carried out by a combination of UPLC separation and ESI-TOF-MS detection. The structures of the designated substances were identified from the protonated-molecular ions [M+H](+) obtained from the TOF-MS measurement. The calibration curves obtained from the peak area ratios of the internal standard (I.S.), i.e., 3-phenyl-1-propylamine, and the designated substances versus the injection amounts showed good linearity. The limits of detection (S/N = 3) and the limits of quantification (S/N = 10) in 0.1 mL of human plasma and urine for the present method were 0.30-150 pmol and 1.0-500 pmol, respectively. Good accuracy and precision (according to intraday and interday assays) were also obtained with the present procedure. This method was applied to analyses of human plasma, urine and real products.

Ultra performance liquid chromatography-tandem mass spectrometry for the determination of 5-nitroimidazoles and their metabolites in egg

Abstract An ultra performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS) procedure was developed for the determination of five 5-nitroimidazoles (dimetridazole, ipronidazole, metronidazole, ronidazole and ternidazole) and three of their metabolites (1-methyl-2-hydroxymethyl-5-nitroimidazole, 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole and 1-methyl-2-(2′-hydroxyisopropyl)-5-nitroimidazole) in egg matrices. Conditions for UPLC separation and electrospray ionization MS/MS in the positive ion mode were optimized. Samples were prepared by liquid-liquid extraction with buffered aqueous 2.5% trichloroacetic acid followed by solid-phase extraction on a Strata-X-C cartridge with reversed-phase and cation-exchange functionalities. The method’s performance was evaluated in accordance with Commission Decision 2002/657/EC, applying the alternative matrix-comprehensive in-house validation approach using specially designed InterVal software. The method was robust against different sample matrix and SPE cartridges, operator change, and changes in sample extract storage. Acceptable apparent recoveries (76 to 109%) were obtained for all analytes. The decision limits (CCα) and detection capabilities (CCα) were within the ranges of 0.19–2.62 and 0.26–4.29 μg kg−1. For all compounds the calibration curve linearity was good with correlation coefficients greater than 0.99. Fifteen eggs and ten whole egg powder samples obtained commercially in Lithuania were analyzed by UPLC-MS/MS; none were found contaminated by 5-nitroimidazoles or their metabolites.