Stable Isotope-dilution Liquid Chromatography-tandem Mass Research Articles

Serum S-adenosylhomocysteine levels are associated with first stroke in Chinese adults with hypertension

BackgroundThe association between S-adenosylhomocysteine (SAH) and stroke has not been confirmed due to the specialized equipment and time requirements necessary for S-adenosylhomocysteine testing. We aimed to explore the association between SAH and stroke. MethodsA nested, case-control study drawn from the China Stroke Primary Prevention Trial of rural adults with hypertension, including 557 first stroke cases and 557 matched controls was conducted. Serum SAH was measured by stable-isotope dilution liquid chromatography-tandem mass spectrometry using 4500MD. Multiple conditional logistic regression models were used to evaluate the association between SAH and first stroke. ResultsIn females, SAH levels were significantly higher in the stroke population than in the control group (16.0 ng/mL vs. 14.6 ng/mL). When SAH was assessed as quartiles, the odds of stroke were 1.78 (95 % CI: 1.02–3.09) in Quartile 2, 1.31 (95 % CI: 0.73–2.33) in Quartile 3, and 1.93 (95 % CI: 1.03–3.62) in Quartile 4, compared to Quartile 1. When Quartiles 2–4 were combined, the adjusted odds ratio of first stroke was 1.64 (95 % CI: 1.03–2.62) compared with Quartile 1. In subgroup analysis, a significant SAH-stroke association was observed in the lower vitamin D3 group (OR = 3.35, 95 % CI:1.72–6.53; P interaction, 0.035). In males, higher levels of SAH were associated with an increased risk of stroke in those under age 60. Compared with the reference group, the adjusted odds ratio of total stroke was 2.40 (95 % CI: 1.02–5.91) in the combined group (Quartile 2–4). In contrast, no significant association between SAH and stroke was found in males aged 60 or older. ConclusionsThis study reveals that SAH is associated with a higher risk of stroke independently of homocysteine, especially in females. SAH may be a second predictor of stroke in the metabolic pathway of methionine, after homocysteine.

Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy

A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

A Rapid, Simple, Trace, Cost-Effective, and High-Throughput Stable Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Method for Serum Methylmalonic Acid Quantification and Its Clinical Applications.

HighlightsWhat are the main findings?An established method for trace, simple, rapid, cheap, sensitive, accurate, robust, and high-throughput for methylmalonic acid quantification.Good chromatographic separation of MMA and its intrinsic isomer and good signals of MMA were achieved using a simple isocratic elution strategy.Materials and reagents that are complex or not always accessible and procedures in previous methods such as derivatization, multistep SPE, incubations, evaporations, drying, or reconstitutions are not required in this MMA quantification method.What is the implication of the main finding?This method is suitable for large-scale MMA testing.Background: Methylmalonic acid (MMA) is an essential indicator of vitamin B12 (VB12) deficiency and inherited metabolic disorders (IMDs). The increasing number of requests for MMA testing call for higher requirements for convenient MMA testing methods. This study aims to develop a convenient quantification method for serum MMA. Methods: The method was established based on the stable isotope-dilution liquid chromatography–tandem mass spectroscopy (ID-LC-MS/MS) technique. The LC-MS/MS parameters and sample preparation were optimized. Specificity, sensitivity, robustness, accuracy, and clinical applicability were validated according to CLSI C62-A guidelines. MMA levels in VB12-sufficient subjects and VB12-deficient subjects were measured. Results: MMA and its intrinsic isomer, i.e., succinic acid (SA), were completely separated. The average slope, intercept, and correlation relationship (R) with 95% confidence intervals, during the two months, were 0.992 (0.926–1.059), −0.004 (−0.012–0.004), and 0.997 (0.995–0.999), respectively. The limit of detection and quantification were <0.058 μmol/L and 0.085 μmol/L, respectively. Intra-run, inter-run, and total imprecisions were 1.42–2.69%, 3.09–5.27%, and 3.22–5.47%, respectively. The mean spiked recoveries at the three levels were 101.51%, 92.40%, and 105.95%, respectively. The IS-corrected matrix effects were small. The VB12-deficient subjects showed higher MMA levels than VB12-sufficient subjects. Conclusions: A convenient LC-MS/MS method for serum MMA measurement was developed and validated, which could be suitable for large-scale MMA testing and evaluating MMA levels in VB12-deficient patients.

Association between serum 5-methyltetrahydrofolate and homocysteine in Chinese hypertensive participants with different MTHFR C677T polymorphisms: a cross-sectional study

Background and aimsClarifying the association between 5-methyltetrahydrofolate and homocysteine and the effect pattern of methylene tetrahydrofolate reductase (MTHFR C677T) may contribute to the management of homocysteine and may serve as a significant reference for a randomized controlled trial of 5-methyltetrahydrofolate intervention. This study aimed to reveal the association between these two biochemical indices.MethodsStudy population was drawn from the baseline data of the China Stroke Primary Prevention Trial (CSPPT), including 2328 hypertensive participants. 5-methyltetrahydrofolate and homocysteine were determined by stable-isotope dilution liquid chromatography-tandem mass spectrometry and automatic clinical analyzers, respectively. MTHFR C677T polymorphisms were detected using TaqMan assay. Multiple linear regression was performed to evaluate the association between serum 5-methyltetrahydrofolate and homocysteine.ResultsThere was a significant inverse association between 5-methyltetrahydrofolate and homocysteine when 5-methyltetrahydrofolate was ≤ 10 ng/mL, and this association was modified by MTHFR C677T (per 1-ng/mL increment; All: β = − 0.50, P < 0.001; CC: β = − 0.14, P = 0.087; CT: β = − 0.20, P = 0.011; TT: β = − 1.19, P < 0.001). Moreover, the decline in trend in genotype TT participants was stronger than in genotype CC participants (P for difference < 0.001) and genotype CT participants (P for difference < 0.001), while there was no significant difference between genotype CC and genotype CT participants (P for difference = 0.757).ConclusionsOur data showed a non-linear association between serum homocysteine and 5-methyltetrahydrofolate among Chinese hypertensive adults, however, it could be inversely linearly fitted when serum 5-methyltetrahydrofolate was ≤ 10 ng/mL, and this association was modified by MTHFR C677T.

Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize.

The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.

Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of Genotoxic Chemicals Revealed by Mass Spectrometry Analysis.

Genotoxic chemicals act by causing DNA damage, which, if left unrepaired, can have deleterious consequences for cell survival. DNA damage response (DDR) gets activated to repair or mitigate the effects of DNA damage. Histone H2AX and H3 phosphorylation biomarkers (γ-H2AX and p-H3) have attracted great attention as they play pivotal roles in the DDR. Simultaneous quantitation of γ-H2AX and p-H3 in exposed cells may monitor the toxicity of genotoxic chemicals and to some extent reflect the subsequent DDR process. Reported here is the first comprehensive characterization of distinct orchestration and dynamic processes on cellular γ-H2AX and p-H3 for two major types of genotoxic chemicals, clastogens and aneugens, by stable isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). We find that clastogens significantly induce an increase in γ-H2AX and a decrease in p-H3; aneugens have no obvious effect on γ-H2AX, whereas induce either an increase or a decrease in p-H3. In addition, the specific profiles of clastogens and aneugens affecting DNA damage may be dynamically observed, which in turn provides insights into the processes involving DNA damage repair as well as transcription. Taken together, these results suggest that robust LC-MS/MS analysis of γ-H2AX and p-H3 can not only quantitatively differentiate mechanistic information on clastogens and aneugens but also dynamically present the detail profiles of DNA damage and repair processes.

Targeted lipidomics profiling of acute arsenic exposure in mice serum by on-line solid-phase extraction stable-isotope dilution liquid chromatography-tandem mass spectrometry.

Lipidomics is the area of study dedicated to the comprehensive analysis and characterization of the functions and metabolism of lipids in biological samples. One of the most comprehensively studied classes of lipids is polyunsaturated fatty acids (PUFAs). Eicosanoids are a series of bioactive lipid mediators that are metabolized from PUFAs and generated majorly from the precursor arachidonic acid. This study identified the profiles of target eicosanoids after acute exposure to arsenic. The principle objective was to determine and validate 10 eicosanoids in mouse serum using on-line solid-phase extraction integrated with liquid chromatography-electrospray tandem mass spectrometry. Intra-day and inter-day repeatability was 82.4-119.2 and 86.7-124.4%, respectively. The limit of detection and limit of quantification were from 0.003 to 0.288ng/mL and from 0.009 to 0.962ng/mL, respectively. The levels of 7 of the 10 eicosanoids-namely 8-isoPGF2α, PGF2α, PGE2, 13(s)-HODE, 15(s)-HETE, 12(s)-HETE, and 5(s)-HETE-in mouse serum significantly and dose-dependently increased after arsenic exposure compared with the levels in the vehicle control group. To our knowledge, this is the first study to quantify eicosanoids in mouse serum. This approach provides simple sample preparation, small sample volumes, and a precise and accurate method for determining changes in the profile of these eicosanoids in mouse serum after acute exposure to arsenic.

A sensitive method to determine melatonin in saliva by automated online in-tube solid-phase microextraction coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry

Melatonin (MLT) plays important roles in regulating the sleep-wake cycle, and has many beneficial effects on health. A simple, rapid, and sensitive method was developed for the determination of MLT in human saliva by automated online in-tube solid-phase microextraction coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry.

Determination of bisphenols in beverages by mixed-mode solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry

Facing growing restrictions on the use of bisphenol A in food contact materials, several bisphenol analogs are arising as major alternatives to replace this chemical in most of its applications. This work reports a simple and robust method based on mixed-mode solid-phase extraction and stable-isotope dilution liquid chromatography–tandem mass spectrometry for the analysis of bisphenol A and its main analogs – bisphenol S, 4,4′-sulfonylbis(2-methylphenol), bisphenol F, bisphenol E, bisphenol B, bisphenol Z, bisphenol AF, bisphenol AP, tetrabromobisphenol A and bisphenol P – in alcoholic and non-alcoholic beverages. Mixed-mode solid-phase extraction, combining cationic exchange and reversed-phase mechanisms, was optimized to provide a selective extraction and purification of the target analytes. Derivatization of bisphenols with pyridine-3-sulfonyl chloride allowed increasing their ionization efficiency by electrospray ionization. Validation of the proposed method was performed in terms of selectivity, matrix effects, linearity, precision, measurement uncertainty, trueness and limits of detection. Satisfactory repeatability and intermediate precision were obtained; the related relative standard deviations were ≤9% and ≤12%, respectively. The relative expanded uncertainty (k=2) was below 20% for all bisphenol analogs and the trueness of the method was demonstrated by recovery experiments. Limits of detection (LOD) ranged from 1.6ngL−1 to 27.9ngL−1 for all compounds. Finally, several canned and non-canned beverages were analyzed to demonstrate the applicability of the method. Only bisphenol A and three bisphenol F isomers were detected in any of the samples. Bisphenol A concentration ranged from <LOD to 1.26±0.09μgL−1, whereas 4,4′-bisphenol F varied from <LOD to 1.00±0.08μgL−1. To the best of our knowledge, 2,2′-bisphenol F and 2,4′-bisphenol F were reported for the first time in beverages, at concentration levels up to 0.12 and 0.51μgL−1, respectively.

Development and validation of a stable-isotope dilution liquid chromatography–tandem mass spectrometry method for the determination of bisphenols in ready-made meals

Due to their growing consumption, ready-made meals are a major dietary component for many people in today's society, representing an important potential route of human exposure to several food contaminants. The recent restrictions in the use of bisphenol A have led the plastic industry to look for alternative chemicals, most of them belonging to the same family of p,p′-bisphenols.The aim of the current work was to develop and validate a method based on stable-isotope dilution liquid chromatography–tandem mass spectrometry for the analysis of bisphenol A and its main analogs – bisphenol S, 4,4′-sulfonylbis(2-methylphenol), bisphenol F, bisphenol E, bisphenol B, bisphenol Z, bisphenol AF, bisphenol AP, tetrabromobisphenol A and bisphenol P – in solid foodstuffs, and particularly in ready-made meals.Extraction was carried out by ultrasound-assisted extraction after sample disruption with sand. A selective solid-phase extraction procedure was then applied to reduce potential matrix interferences. Derivatization of bisphenols with pyridine-3-sulfonyl chloride increased their ionization efficiency by electrospray ionization. Validation of the proposed method was performed in terms of selectivity, matrix effects, linearity, precision, measurement uncertainty, trueness and limits of detection. Satisfactory repeatability and intermediate precision were obtained; the related relative standard deviations were ≤7.8% and ≤10%, respectively. The relative expanded uncertainty (k=2) was below 17% for all bisphenol analogs and the trueness of the method was demonstrated by spike recovery experiments. Low limits of detection, in the range from 0.025μgkg−1 to 0.140μgkg−1, were obtained for all compounds. To demonstrate the applicability of the proposed method, it was eventually applied to several ready-made meals purchased from different supermarkets in Belgium.

Determination of regorafenib (BAY 73-4506) and its major human metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in human plasma by stable-isotope dilution liquid chromatography-tandem mass spectrometry.

Regorafenib (BAY 73-4506, commercial name Stivarga(®)) is an oral multikinase inhibitor developed by Bayer Pharma AG (Germany) that targets angiogenic, stromal and oncogenic receptor tyrosine kinases. An isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the simultaneous determination of regorafenib and its two major metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in lithium-heparinized human plasma. Analysis was performed after protein precipitation on a triple-quadrupole tandem mass spectrometer. The validated concentration range was from 2.00 (lower limit of quantitation) to 2000 µg/l for all the analytes. Selectivity and specificity in the presence of four potential comedications were demonstrated. Interassay accuracy and precision coefficient of variation ranged from 91.2% (at LLOQ of M-5) to 105% and 3.59 to 14.1%, respectively. The method proved to be selective, specific, sufficiently sensitive, highly reproducible and robust for the analysis of large numbers of samples. The method performance met current guidelines on bioanalytical method validation.

Determination of cyclophosphamide and ifosfamide in sewage effluent by stable isotope-dilution liquid chromatography–tandem mass spectrometry

A reliable and specific method was developed for the determination of the cytotoxic drugs cyclophosphamide and ifosfamide in sewage effluent. The most successful combination was found to be Strata-X solid-phase extraction followed by Florisil ® clean-up with analysis by liquid chromatography–tandem mass spectrometry. Quantification by internal standardisation was achieved using custom synthesised d4-cyclophophosphamide. The mass spectrometer was operated in highly selective reaction monitoring (HSRM) mode, which significantly reduced matrix noise and improved sensitivity. Although it suffered from some ionisation suppression, electrospray ionisation (ESI) was found to give an order of magnitude better sensitivity in terms of limit of detection than atmospheric pressure chemical ionisation (APCI). Using final effluent from two different sewage treatment plants, the method was validated following official European guidelines and shown to be a high performance tool for routine analysis at the sub-nanogram per litre level. Depending on the matrix, the limit of detection for cyclophosphamide was between 0.03 ng/L and 0.12 ng/L and for ifosfamide between 0.05 ng/L and 0.09 ng/L. For cyclophosphamide the accuracy and precision, tested at 1.7 ng/L, were 98–109% and ≤13%, CV respectively. For ifosfamide the accuracy and precision, tested at 1.1 ng/L, were 98–113% and ≤15% CV, respectively. Depending on the sample matrix the absolute recovery of the internal standard was between 57% and 70%. The method was tested by analysis of spot samples taken from the final effluent discharges of two sewage treatment plants; the first using a conventional trickling filter treatment process and second employing activated sludge followed by ultra violet treatment. Cyclophosphamide was detected at 0.19 ng/L at the first plant and at the second detected at 3.7 ng/L and 3.5 ng/L, before and after the UV treatment process; ifosfamide was not detectable at either plant.

Serum testosterone levels measured by isotope dilution-liquid chromatography–tandem mass spectrometry in postmenopausal women versus those in women who underwent bilateral oophorectomy

Differentiation between subtle changes in low serum testosterone concentrations, common in women and children, is not possible with current commercially available assays. The objectives of the study were to develop a method based on stable isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with adequate sensitivity and specificity and to investigate the applicability of this assay in serum samples from pre- and postmenopausal women. For 16 women, testosterone levels were measured in blood samples drawn two years before and after physiological menopause, and for eight women in samples drawn before and after bilateral oophorectomy. Testosterone was extracted from serum, derivatized and analysed on an LC-MS/MS. The developed ID-LC-MS/MS method allowed for specific and reproducible measurement of testosterone. Comparison with stable isotope dilution-gas chromatography coupled to mass spectrometry detection by Deming regression analysis gave a slope of 1.025 and an intercept of 0.055 nmol/L (r = 0.9998). A significant decrease was found in testosterone concentrations before and after bilateral oophorectomy (P = 0.02), whereas no significant difference was found before and after natural menopause (P = 0.4). The ID-LC-MS/MS assay measures serum testosterone with acceptable accuracy and is useful in female samples, supporting the conclusion that the postmenopausal ovary contributes to circulating testosterone. To our knowledge, our analytical method compares favourably to similar published methods in terms of sensitivity. The sensitivity and specificity of this method comply with the reference method for measurement of testosterone in serum samples of women, children and men suffering from hypogonadism and can also be used for men with testosterone in the reference range.

Testosterone Measurement by Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry: Validation of a Method for Routine Clinical Practice

Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory. We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h. Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.

Quantitative Measurement of Porphobilinogen in Urine by Stable-Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.