Ultra-performance Liquid Chromatography-tandem Research Articles

Determination of bromate in drinking water by ultraperformance liquid chromatography–tandem mass spectrometry

Bromate is a byproduct formed as a result of disinfection of bromide-containing source water with ozone or hypochlorite. The International Agency for Research on Cancer has recognized bromate as a possible human carcinogen, thus it is essential to determine in drinking water. Present work highlights a development of sensitive and fast analytical method for bromate determination in drinking water by using ultraperformance liquid chromatography-tandem mass spectrometry. The quality parameters of the developed method were established, obtaining very low limit of detection (0.01 ng/mL), repeatability and reproducibility have been found to be less than 3% in terms of relative standard deviation when analyzing a bromate standard at 0.05 μg/mL with 0.4 min analysis time. Developed method was applied for the analysis of metropolitan and bottled water from Saudi Arabia; 22 samples have been analyzed. Bromate was detected in the metropolitan water samples (from desalinization source) at concentrations ranging between 3.43 and 75.04 ng/mL and in the bottled water samples at concentrations ranging between 2.07 and 21.90 ng/mL. Moreover, in comparison to established analytical methods such as liquid chromatography-tandem mass spectrometry, the proposed method was found to be very sensitive, selective and rapid for the routine analysis of bromate at low level in drinking water.

Ultra Performance Liquid Chromatography–Tandem Quadrupole Mass Spectrometry Profiling of Anthocyanins and Flavonols in Cowpea (Vigna unguiculata) of Varying Genotypes

The structure of flavonoids in food plants affects bioactivity and important nutritional attributes, like micronutrient bioavailability. This study investigated flavonol and anthocyanin compositions of cowpea (Vigna unguiculata) of varying genotypes. Black, red, green, white, light brown, and golden brown cowpea phenotypes were analyzed for anthocyanins and flavonols using ultra performance liquid chromatography-tandem quadrupole mass spectrometry. Eight anthocyanins and 23 flavonols (15 newly identified in cowpea) were characterized. Mono-, di-, and tri(acyl)glycosides of quercetin were predominant in most phenotypes; myricetin and kaempferol glycosides were present only in specific phenotypes. The red phenotypes had the highest flavonol content (880-1060 μg/g), whereas green and white phenotypes had the lowest (270-350 μg/g). Only black (1676-2094 μg/g) and green (875 μg/g) phenotypes had anthocyanins, predominantly delphinidin and cyanidin 3-O-glucosides. Cowpea phenotype influenced the type and amount of flavonoids accumulated in the seed; this may have implications in selecting varieties for nutrition and health applications.

Establishment and preliminary clinical application of serum bile acid spectrum analysis by ultra high performance liquid chromatography-tandem mass spectrometry

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detecting the bile acid expression in serum and to explore the relationship between the bile acid spectrum and the obesity. Methods After pre-treatment through solid phase extraction, serum samples were chromatographed by gradient elution on an UPLC HSS T3 column, and then mass spectrometric analysis of bile acid was performed by multiple reaction monitoring (MRM) mode with negative electrospray ionization (ESI).And its methodology performance, including specificity, linearity, sensitivity, imprecision and recovery, were verified according to the guideline of State Food and Drug Administration (SFDA).Furthermore，serum samples from 10 simple obesity subjects and 10 healthy controls were analyzed with this established method, and rank sum test was used for statistical analysis. Results This established UPLC-MS/MS method could simultaneously quantify 14 bile acid subgroups in serum sample, its analytical linear range was 10-20 000 nmol/L.For different bile acid subgroups, the limit of detection (LOD) was 0.02-7.90 nmol/L, limit of quantitation (LOQ) was 0.07-44.20 nmol/L; and its within-day coefficient of variation (CV) was 0.35%-12.41%, between-day CV was 1.11%-13.04%; the relative recovery ratio of this method was 89.8%-114.6%.Some differences were observed in chromatogram between obesity and control subjects, and both free bile acid and conjugated bile acid concentrations in obesity subjects ［0.49 （0.45-1.90）, 1.44（0.84-3.72） μmol/L］ were lower than them in control subjects ［0.98（0.53-3.06）, 1.99（0.67-2.88） μmol/L］, but the difference was not significant (Z=-0.958, -0.801, P>0.05). Conclusions This established UPLC-MS/MS method can efficiently differentiate and quantify 14 bile acid subgroups, and is characterized with broad analytical measurement range, good analytical sensitivity and precision.This method can be applied for the free and conjugated bile acid analysis in clinical specimens.(Chin J Lab Med, 2012，35：253-256) Key words: Obesity; Bile acids and salts; Chromatography; high pressure liquid; Tandem mass spectrometry

Measurement of 1- and 3-methylhistidine in human urine by ultra performance liquid chromatography–tandem mass spectrometry

BackgroundDetermination of 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) is important to monitor muscle protein catabolism. Here, an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of 1-MH and 3-MH in human urine is described. MethodsUrine samples were prepared by dilution with water after addition of isotopic internal standard. The samples were chromatographed on a SB-aq (2.1×50mm, 1.8μm) column with acetonitrile −0.1% formic acid in water (2:98, v/v) as mobile phase. Mass spectrometric detection was performed on a triple quadrupole mass spectrometer using positive electrospray ionization (ESI). 1-MH and 3-MH were monitored by the following transitions: 1-MH, m/z 170.1→ m/z 126.1; 3-MH, m/z 170.1→ m/z 124.1. ResultsFor 1-MH and 3-MH, calibration curves were linear over the concentration range of 5–500nmol/ml. The lower limit of quantification was 5nmol/ml. The accuracy was within 85%–115% and precision was <15%. 1-MH and 3-MH were proved to be stable under different storage and processing conditions. In addition, the detection was independent of matrix effect. ConclusionThis rapid and specific UPLC/MS-MS method is suitable for the determination of urinary 1-MH and 3-MH.

Plasma appearance and disappearance of an oral dose of 25-hydroxyvitamin D2in healthy adults

25-Hydroxyvitamin D (25(OH)D) half-life is a potential biomarker for investigating vitamin D metabolism and requirements. We performed a pilot study to assess the approach and practical feasibility of measuring 25(OH)D half-life after an oral dose. A total of twelve healthy Gambian men aged 18–23 years were divided into two groups to investigate the rate and timing of (1) absorption and (2) plasma disappearance after an 80 nmol oral dose of 25(OH)D2. Fasting blood samples were collected at baseline and, in the first group, every 2 h post-dose for 12 h, at 24 h, 48 h and on day 15. In the second group, fasting blood samples were collected on days 3, 4, 5, 6, 9, 12, 15, 18 and 21. Urine was collected for 2 h after the first morning void at baseline and on day 15. 25(OH)D2 plasma concentration was measured by ultra-performance liquid chromatography-tandem MS/MS and corrected for baseline. Biomarkers of vitamin D, Ca and P metabolism were measured at baseline and on day 15. The peak plasma concentration of 25(OH)D2 was 9·6 (sd 0·9) nmol/l at 4·4 (sd 1·8) h. The terminal slope of 25(OH)D2 disappearance was identified to commence from day 6. The terminal half-life of plasma 25(OH)D2 was 13·4 (sd 2·7) d. There were no significant differences in plasma 25(OH)D3, total 1,25(OH)2D, parathyroid hormone, P, Ca and ionised Ca and urinary Ca and P between baseline and day 15 and between the two groups. The present study provides data on the plasma response to oral 25(OH)D2 that will underpin and contribute to the further development of studies to investigate 25(OH)D half-life.

Determination of sitagliptin phosphate in rat plasma by ultra high performance liquid chromatography-tandem mass spectrometry

An ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of sitagliptin phosphate in rat plasma was established. The blank rat plasma sample added with sitagliptin phosphate and the internal standard (fluoxetine) standard solution were prepared. Methanol was added in the sample for the deproteinization. Then the sample was vortex-mixed and centrifuged. The clear supernatant was used for the analysis of UPLC-MS/MS. A Thermo Hypersil Gold C18 column (50 mm x 2.1 mm, 1.9 microm) was employed with a guard column of Phenomenex Security Guard C18 column (4 mm x 3.0 mm), and the column temperature was set at 35 degrees C. The gradient elution of acetonitrile and water (containing 0.05% (v/v) formic acid) as mobile phases was performed at a flow rate of 200 microL/min, and a rapid separation was completed in 5 min. The electrospray was operated in the positive ionization mode and the sitagliptin phosphate and fluoxetine were identified by selected reaction monitoring (SRM) mode, and the monitoring ions of them were m/z 408.0 --> 235.0 and m/z 310.0 --> 148.0, respectively, which were used to qualify and quantity the targets by the method of matrix-matched standard solution. The calibration curve showed good linearity within the concentrations of 1 to 1 000 microg/L (r = 0.999 1); the limit of detection was 0.2 microg/L. The mean recoveries were from 85% to 115% at the spiked levels of 5, 50 and 500 microg/L; the relative standard deviations (RSDs) of intra- and inter-day of variation were both less than 15%, which can meet the determination requirements of biological samples. Then the method was initially used for the determination of sitagliptin phosphate in SD rat plasma after the administration of a single intravenous injection dose of sitagliptin phosphate. The method is rapid, sensitive, convenient and reproducible in the determination of sitagliptin phosphate, and can be used for the pharmacokinetics research of sitagliptin phosphate in plasma.

Rapid Detection of the Residual Kryptofix 2.2.2 Levels in [18F]-Labeled Radiopharmaceuticals by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

A fast and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method was developed and validated for determination of the residual levels of Kryptofix 2.2.2 (K222) in [18F]-labeled radiopharmaceuticals. The analytical time was only 3 min, and the injection volume was 5 μL. An electrospray ionization source was used in the positive mode (ESI+) for UPLC/MS/MS. The analytical measurements were performed in the multiple reaction monitoring (MRM) mode. The calibration curve at the spiked concentrations of 2–500 ng/mL for K222 showed good linearity. The intra- and inter-day precisions were not more than 5%. The accuracy satisfied the requirement of quality control analysis, the recoveries were found to be 80–120%. This method was successfully applied to detect the residue of K222 in [18F]-fluorodeoxyglucose [(18F)FDG], [18F]-fluoromisonizole[(18F)FMISO], 3′-deoxy-3′-[18F]-fluorothymidine [(18F)FLT], and two new [18F]-labeled radiopharmaceuticals 4-[-(2-[18F]fluoroethoxy) methyl]-1-[2-(2-methyl-5-nitro-1H- imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as 18F-BNU-1) and 4-[-(2-[18F] fluoroethoxy) methyl]-1-[2-(2-nitro-1H-imidazol-1-yl) ethyl]-1H-1,2,3-triazole (named as 18F-BNU-2) produced in our lab.

An ultraperformance liquid chromatography–tandem mass spectrometry method for determination of anastrozole in human plasma and its application to a pharmacokinetic study

A fast, selective and sensitive ultraperformance liquid chromatography-tandem mass spectrometry method was developed for determination and pharmacokinetic study of anastrozole in human plasma. Plasma sample pretreatment involved a one-step extraction with diethyl ether of 500 µL plasma. The chromatographic separation was carried out on an Acquity UPLC(TM) BEH C(18) column with a mobile phase consisting of methanol-10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.30 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive mode. A high throughput was achieved with a run time of 1.5 min per sample. The standard curve for anastrozole was linear (r(2) ≥ 0.99) over the concentration range of 0.0550-27.5 ng/mL with a lower limit of quantification of 0.0550 ng/mL. The intra- and inter-day precision (relative standard deviation) values were not higher than 14% and the accuracy (relative error) was within ±3.2% at three quality control levels. This simple, fast and highly sensitive method was fully validated and successfully applied to a clinical pharmacokinetic study of anastrozole in healthy volunteers after oral administration.

Determination of marker residue of Olaquindox in fish tissue by ultra performance liquid chromatography–tandem mass spectrometry

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is the last major remaining detectable metabolite of Olaquindox in animal tissue. A rapid, sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the detection and quantification of MQCA in fish tissue using deuterated quinoxaline-2-carboxylic acid (d(4)-QCA) as internal standard. Various parameters affecting sample preparation, LC separation and MS/MS detection were investigated, and the optimal conditions concerned were determined. Fish tissue samples were subject to hydrochloric acid hydrolysis followed by Oasis MAX solid-phase extraction clean-up; analysis was performed using UPLC coupled to electrospray MS/MS. The chromatographic separation was achieved in less than 5 min. The limit of detection and the limit of quantification were 0.1 and 0.25 ng/g, respectively. The average recoveries of MQCA, spiked at levels of 0.25-50.0 ng/g, were from 92.7 to 104.3%. The relative standard deviation values were <6%. The validated method was successfully applied to analyze 60 batch samples collected from the local market.

Simultaneous Determination of Testosterone and Testosterone Enanthate in Equine Plasma by UHPLC-MS-MS

Testosterone and testosterone enanthate are performance-enhancing substances that are banned in racehorses competing in the State of Pennsylvania (PA). A tolerance concentration of 2,000 pg mL−1 plasma has been established for testosterone in intact colts and stallions at the time they are competing in PA. Testosterone enanthate is a precursor of testosterone and can be used to boost plasma testosterone concentration above natural, age and seasonally variable plasma concentration. To control abuse, a verifiable method for rapid determination of both substances in equine plasma was needed. For this reason, an ultra high performance liquid chromatography-tandem mass spectrometry method for high-throughput analysis of both analytes in equine plasma was developed. Analytes were recovered from plasma by liquid–liquid extraction using mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v), separated on a C18 sub-2 μm column and detected on a triple quadrupole mass spectrometer using positive electrospray ionization mode with selected reaction monitoring scan. SRM ion transitions of m/z 289 → m/z 97, m/z 289 → m/z 109, m/z 289 → m/z 79 were used for testosterone identification while m/z 401 → m/z 253, m/z 401 → m/z 271, m/z 401 → m/z 97 were employed for testosterone enanthate. Retention time and product ion intensity ratio were used as confirmation criteria to ascertain the presence of both analytes in equine plasma. The limits of detection, quantification and confirmation were 50 pg 0.5 mL−1, 100 pg 0.5 mL−1 and 250 pg 0.5 mL−1, respectively for both analytes. The method was validated for recovery efficiency, sensitivity, matrix effect, linearity, precision and accuracy. This method is routinely used in the PA program for androgenic anabolic steroids doping control in racehorses and in the on-going testosterone enanthate pharmacokinetics study. The method is defensible, fast, selective, specific and reproducibly reliable.