Dilution Liquid Chromatography Tandem Mass Research Articles

Preparation of reference materials of total thyroxine

Objective To prepare the serum reference materials for total thyroxine. Methods Individual blood samples were collected from 13 healthy donors (7 males and 6 females) aged from 20 to 50 years old, and the sera were separated and mixed into 4 serum pools according to the concentration of thyroxine. The materials were tested for homogeneity and stability using routine methods. The method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) was used to determine the concentration of thyroxine. The candidate reference materials were also measured by four conventional methods to analyze the commutability of the materials. Results It showed that the four candidate reference materials were homogeneous and commutable in four conventional methods and they were tested to be stable for at least 1 year at -70 ℃ using the isochronous stability study. The certified values (reference value ± expanded uncertainty, nmol/L) were: 75.9±1.8, 105.3±2.2, 114.7±2.1 and 187.4±2.9. Conclusions Certified reference materials for serum thyroxine have been prepared. These materials have been approved to be the Certificate Reference Materials of GBW 09127, GBW 09128, GBW 09129 and GBW 09130.(Chin J Lab Med, 2016, 39: 40-44) Key words: Thyroxine; Chromatograhy, liquid; Tandem mass spectrometry; Reference standards

Occurrence and daily variability of pharmaceuticals and personal care products in swimming pools.

The aim of this research was to investigate the presence and daily variability of pharmaceuticals and personal care products (PPCPs) in public swimming pools. Various types of public swimming pool water were analysed, taken from freshwater indoor swimming pools, outdoor swimming pools, spa pools and seawater swimming pools. Swimming pool water samples were analysed for 30 PPCPs using solid phase extraction (SPE) followed by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). All PPCPs were below quantification limits in seawater pools. However, caffeine was detected in 12 chlorinated swimming pools at concentrations up to 1540 ng/L and ibuprofen was observed in 7 chlorinated pools at concentrations up to 83 ng/L. Caffeine and ibuprofen concentrations were below quantification limits in all fill water samples, eliminating this as their source in swimming pools. High variations in caffeine concentrations monitored throughout the day roughly reflect bather loads in swimming pools. Future monitoring of these compounds may assist in evaluating what portion of organic matter measured in swimming pools may come from human excretions.

동위원소희석 액체크로마토그래피 질량분석법에 의한 분유 내 콜레스테롤의 정량

분유 내 콜레스테롤을 정량분석하기 위한 일차분석법으로 동위원소희석 질량분석법을 개발하였다. 내부표준물질로 콜레스테롤-d₄를 사용하여 분유에 첨가하였다. 분유에 지방산과 에스테르형태로 결합되어 있는 콜레스테롤을 자유 콜레스테롤로 바꾸어주기 위해 비누화과정을 수행하였다. 비누화과정은 반응온도, 반응시간, 사용한 KOH의 농도에 따라서 최적화하였으며, 그 결과로 분유시료 0.1 g에 대해 70 ℃에서 180 분 동안 8 M의 KOH 0.8 mL를 첨가하여 반응을 진행시키는 최적화조건을 확립하였다. 이와 같은 조건으로 실험을 진행하여 재현성은 0.23%, 확장불확도는 95%의 신뢰범위에서 1.9%로 추정되었다. 확립된 동위원소희석 질량분석법의 유효화를 위해 분유내에서 콜레스테롤의 인증값을 가지는 NIST SRM을 측정하였고 이 결과가 인증값과 불확도 범위내에서 일치하는 것을 확인하였다. An isotope dilution liquid chromatography tandem mass spectrometry was developed as a primary method for the quantitative analysis of cholesterol in infant formula. Cholesterol-d₄ was used as an internal standard and spiked into the infant formula sample. In order to release cholesterol out of cholesteryl ester, which is cholesterol bound to fatty acids in infant formula, saponification was carried out. Saponification conditions were optimized with heating temperature, reaction time and the concentration of KOH. The optimum conditions were as follows; heating temperature was 70 ℃, reaction time was 180 min and the concentration of KOH was 0.8 mL of 8 M KOH for about 0.1 g infant formula sample. Extraction of cholesterol out of sample solution was carried out with hexane uisng liquid-liquid extraction. Chromatographic analysis was carried out using Phenomenex Kinetex C₁₈ column. Mobile phase was 0.1% acetic acid in methanol/water (v/v, 99/1) and flow rate was 0.3 mL/min. Cholesterol and cholesterol-d₄ were monitored at mass transfer m/z 369/259 and 373/263 respectively. Reproducibility of the method was evaluated to be 0.23% of the measurement result. The expanded uncertainty of the measurement result of cholesterol in infant formula was approximately 1.9% at a 95% confidence level. NIST standard reference material having certified values of cholesterol in infant formula, was analyzed in order to verify this method. The ID-LC/MS/MS results were well agreed with the certified values of NIST SRM within the uncertainty.

Quantification of 16 polycyclic aromatic hydrocarbons in cigarette smoke condensate using stable isotope dilution liquid chromatography with atmospheric-pressure photoionization tandem mass spectrometry.

A stable isotope dilution liquid chromatography tandem mass spectrometry method for the analysis of 16 polycyclic aromatic hydrocarbons in cigarette smoke condensate was developed and validated. Compared with previously reported methods, this method has lower limits of detection (0.04-1.35 ng/cig). Additionally, the proposed method saves time, reduces the number of separation steps, and reduces the quantity of solvent needed. The new method was applied to evaluate polycyclic aromatic hydrocarbon content in 213 commercially available cigarettes in China, under the International Standardization Organization smoking regime and the Health Canadian intense smoking regime. The results showed that the total polycyclic aromatic hydrocarbon content was more than two times higher in samples from the Health Canadian intense smoking regime than in samples from the International Standardization Organization smoking regime (1189.23 versus 2859.50 ng/cig, p < 0.05). Meanwhile, the concentration of individual polycyclic aromatic hydrocarbons (and total polycyclic aromatic hydrocarbons) increased with labeled tar content in both of the tested smoking regimes. There was a positive correlation between total polycyclic aromatic hydrocarbons under the International Standardization Organization smoking regime with that under the Health Canadian intense smoking regime. The proposed liquid chromatography tandem mass spectrometry method is satisfactory for the rapid, sensitive, and accurate quantitative evaluation of polycyclic aromatic hydrocarbon content in cigarette smoke condensate, and it can be applied to assess potential health risks from smoking.

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.

Trueness evaluation of 13 routine measurements for serum uric acid

Objective To evaluate the trueness of 13 routine measurements for serum uric acid and the role of reference method in improving harmonization and trueness among routine measurement systems. Methods The research is related to the reagent evaluation. Usingisotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method as the comparison method, Wako, Sekisui, DiaSys, Maker, Dirui, Leadman, BSBE, Biosina, Mindray, MedicalSystem, LongMarch, and Kehua 13 kinds of uric acid kits were chosen as the evaluation methods with Hitachi 7170A as the analyzer. serum uric acid in 40 fresh frozen serawere collected from clinical laboratory of Beijing hospital in 2014, coveringboth physiological and pathological status (80-940 μmol/L). 19 kinds of prepared materials and the 40 fronzen sera were measuredby comparison method and evaluation methods and linear regression analysis was made for the results. The performance of evaluation methods was revealed and recalibration was performed on every evaluation methodby the linear regression equation. The variation of percent bias(%)of the uric acid values in 19 preparation materials was compared. Results All test methods demonstrated good precision (CV 0.998, P<0.01) with the comparison method when measuring uric acid values in 40 fresh frozen sera The meanpercent bias was 0.17% (-3.06% - 7.31%). After recalibration, 4 of 19 samples with no matrix effect values percent bias reduced and met the demands of quality (<4.8%)induced from biological variation. Conclusion All test methods demonstrated good trueness and their calibration traceability was verified. Recalibration using reference method or standard reference materials contributes to harmonization among methods.(Chin J Lab Med, 2015, 38: 609-612) Key words: Uric acid; Diagnostic tests, routine; Reference values; Reagent kits, diagnosis; Reproducibility of results; Bias (epidemiology)

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.

The commutability of reference materials for serum glucose measurements

Objective To evaluate the commutability of certified reference materials, external quality assessment program materials and calibrators for serum glucose measurements which were performed in 24 routine measurement procedures. Methods 35 fresh patient specimens and some reference materials were analyzed by isotope dilution liquid chromatography tandem mass spectrometry (as the comparative method) and 24 routine measurement procedures (as the evaluated methods). The relationships between the results from the evaluated method and the comparative methods were evaluated to identify the commutability. Results It showed that 5 certified reference materials, 2 trueness verification materials, and 5 calibrators were commutable in all 24 routine measurement procedures. The other samples were displayed the presence of commutability issue in different degrees. Conclusion It is important to pay more attention to the problems brought by commutability of reference materials in clinical laboratory.(Chin J Lab Med, 2015, 38: 296-300) Key words: Blood glucose; Reference standards; Reference values

Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

Serum total thyroxine measured by isotope dilution liquid chromatography tandem mass spectrometry

Objective An isotope dilution liquid chromatography tandem mass spectrometric (ID-LC/MS/MS) method was developed for serum total thyroxine. Methods Serum samples were sampled by weight and the 13C6-thyroxine internal standards were added volumetrically using automated dilutors, followed by equilibration, protein precipitation, and cation exchang solid-phase extractions(SPE). After SPE, the eluates were evaporated to dryness under nitrogen and then the evaporated residues were reconstituted to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis.Seven kinds of serum pools were used to analyze the precision of the method.The analytical recovery, the limit of detection(signal to noise ratio, S/N ratio 3∶1) and the limit of quantification (S/N ratio 10∶1)for thyroxine were calculated.Also, in order to evaluate the accuracy of this method, the candidate reference materials from German Societies for Clinical Chemistry were analyzed. Results The within-run and total coefficients of variation were: 0.64%(0.35%-0.89 %)and 0.83%(0.57%-1.37 %), respectively.The analytical recoveries ranged from 99.6% to 100.7%.The limit of detection was 0.12 nmol/L and the limit of quantification was 0.41 nmol/L for thyroxine in human serum.The results of analyzing the reference material of CRM21201 and CRM21202 showed biases of -0.30% (ranged from -0.13% to 0.73%). Conclusions A highly precise and accurate method for measuring serum total thyroxine has been developed by ID-LC/MS/MS. The method is less sample volume demand and less time-consuming and may be used as a candidate reference method. Key words: Thyroxine; Isotopes; Indicators and reagents; Chromatography, liquid; Tandem mass spectrometry

Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography–tandem mass spectrometry

An ultrasensitive stable isotope dilution liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1fg on column, and was 10fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200pg/mL (5–2000pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7pg/mL) were very similar to those obtained by highly sensitive gas chromatography–mass spectrometry (GC–MS) methodology. However, the level obtained in serum from older men (mean 9.5pg/mL) was lower than has been reported previously by both GC–MS and LC–MS procedures. The total (unconjugated+conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases.

Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization

BackgroundA high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100μl human serum for each measurement (triplicates consume 300μl) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform. MethodsSteroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women. ResultsThe LOD, LOQ, sLOQ was (0.1pg, 0.3pg, 1pg/ml) for estrone, (0.3pg, 1pg, 3pg/ml) for estradiol, and (0.3pg, 1pg, 30pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels. ConclusionsWe have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry.

Bioaccumulation and trophic dilution of human pharmaceuticals across trophic positions of an effluent-dependent wadeable stream.

Though pharmaceuticals are increasingly observed in a variety of organisms from coastal and inland aquatic systems, trophic transfer of pharmaceuticals in aquatic food webs have not been reported. In this study, bioaccumulation of select pharmaceuticals was investigated in a lower order effluent-dependent stream in central Texas, USA, using isotope dilution liquid chromatography-tandem mass spectrometry (MS). A fish plasma model, initially developed from laboratory studies, was tested to examine observed versus predicted internal dose of select pharmaceuticals. Pharmaceuticals accumulated to higher concentrations in invertebrates relative to fish; elevated concentrations of the antidepressant sertraline and its primary metabolite desmethylsertraline were observed in the Asian clam, Corbicula fluminea, and two unionid mussel species. Trophic positions were determined from stable isotopes (δ(15)N and δ(13)C) collected by isotope ratio-MS; a Bayesian mixing model was then used to estimate diet contributions towards top fish predators. Because diphenhydramine and carbamazepine were the only target compounds detected in all species examined, trophic magnification factors (TMFs) were derived to evaluate potential trophic transfer of both compounds. TMFs for diphenhydramine (0.38) and carbamazepine (1.17) indicated neither compound experienced trophic magnification, which suggests that inhalational and not dietary exposure represented the primary route of uptake by fish in this effluent-dependent stream.

Determination of the maleic acid in rat urine and serum samples by isotope dilution–liquid chromatography–tandem mass spectrometry with on-line solid phase extraction

A rapid and simple on-line solid-phase extraction coupled with isotope dilution–liquid chromatography–tandem mass spectrometry (SPE–ID–LC–MS/MS) method was developed to quantitate maleic acid in serum and urine of SpragueDawley (SD) rats. The aforementioned biological samples were spiked with 13C2-maleic acid, vigorously vortexed, added with acetonitrile to precipitate proteins, and then injected into the on-line SPE–LC–MS/MS system for quantification. Upon validation, this method demonstrated excellent feasibility and sensitivity: calibration curves for maleic acid in serum and urine display excellent linearity with the coefficient of determination (R2) greater than 0.999; the limits of detection and quantitation (LOD and LOQ) for maleic acid were determined at 0.2 and 0.5μgL−1, respectively. Additionally, intra-day accuracy for maleic acid in serum and urine samples ranged from 94.0% to 100.2% and 101.3% to 104.4%, respectively. Furthermore, inter-day accuracy ranged from 93.6% to 101.0% and from 102.3% to 111.4% in serum and urine samples, respectively. Intra-day precision %RSD of maleic acid in serum and urine samples was 13.8% or less, whereas the inter-day precision was 6.1% or less. The matrix effects were not found to be statistically significant (p=0.9145 and p=0.5378, correspondingly) based on the calculations of recovery functions. The collected serum and urine samples were analyzed using SPE–ID–LC–MS/MS. Our results reveal trace levels of maleic acid in the control rats, demonstrating that this method is capable of analyzing background levels of contaminants in biofluids with excellent sensitivity and specificity at part-per-billion levels concentrations in complex matrices.

Simultaneous determination of seven β2-agonists in human and bovine urine by isotope dilution liquid chromatography–tandem mass spectrometry using compound-specific minimally 13C-labelled analogues

Seven β2-agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC–ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally 13C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg−1 levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg−1 ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg−1 for all compounds, except to salbutamol in bovine urine which was of 0.029ngg−1. The use of compound-specific isotopically labelled analogues minimally labelled in 13C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of β2-agonists in human and bovine urine.

Human aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons.

Aldo-keto reductases (AKRs) are promiscuous NAD(P)(H) dependent oxidoreductases implicated in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). These enzymes catalyze the oxidation of non-K-region trans-dihydrodiols to the corresponding o-quinones with the concomitant production of reactive oxygen species (ROS). The PAH o-quinones are Michael acceptors and can form adducts but are also redox-active and enter into futile redox cycles to amplify ROS formation. Evidence exists to support this metabolic pathway in humans. The human recombinant AKR1A1 and AKR1C1–AKR1C4 enzymes all catalyze the oxidation of PAH trans-dihydrodiols to PAH o-quinones. Many human AKRs also catalyze the NADPH-dependent reduction of the o-quinone products to air-sensitive catechols, exacerbating ROS formation. Moreover, this pathway of PAH activation occurs in a panel of human lung cell lines, resulting in the production of ROS and oxidative DNA damage in the form of 8-oxo-2′-deoxyguanosine. Using stable-isotope dilution liquid chromatography tandem mass spectrometry, this pathway of benzo[a]pyrene (B[a]P) metabolism was found to contribute equally with the diol-epoxide pathway to the activation of this human carcinogen in human lung cells. Evaluation of the mutagenicity of anti-B[a]P-diol epoxide with B[a]P-7,8-dione on p53 showed that the o-quinone produced by AKRs was the more potent mutagen, provided that it was permitted to redox cycle, and that the mutations observed were G to T transversions, reminiscent of those observed in human lung cancer. It is concluded that there is sufficient evidence to support the role of human AKRs in the metabolic activation of PAH in human lung cell lines and that they may contribute to the causation of human lung cancer.

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.

Association of branched-chain amino acids with carotid intima-media thickness and coronary artery disease risk factors.

BackgroundRecent studies have determined that branched-chain (BCAAs) and aromatic (AAAs) amino acids are strongly correlated with obesity and atherogenic dyslipidemia and are strong predictors of diabetes. However, it is not clear if these amino acids are capable of identifying subjects with coronary artery disease (CAD), particularly with subclinical atherosclerosis who are at risk of developing CAD.MethodsFour hundred and seventy two Chinese subjects (272 males and 200 females, 42–97 y of age) undergoing physical exams were recruited at random for participation in the cross-sectional study. Serum BCAAs and AAAs were measured using our previously reported isotope dilution liquid chromatography tandem mass spectrometry method. Bilateral B-mode carotid artery images for carotid intima-media thickness (cIMT) were acquired at end diastole and cIMT values more than 0.9 mm were categorized as increased. Correlations of BCAAs with cIMT and other CAD risk factors were analyzed.ResultsBCAAs and AAAs were significantly and positively associated with risk factors of CAD, e.g., cIMT, BMI, waist circumference, blood pressure, fasting blood glucose, TG, apoB, apoB/apoAI ratio, apoCII, apoCIII and hsCRP, and were significantly and negatively associated with HDL-C and apoAI. Stepwise multiple linear regression analysis revealed that age (β = 0.175, P<0.001), log BCAA (β = 0.147, P<0.001) and systolic blood pressure (β = 0.141, P = 0.012) were positively and independently associated with cIMT. In the logistic regression model, the most and only powerful laboratory factor correlated with increased cIMT was BCAA (the odds ratio of the fourth quartile compared to the first quartile was 2.679; P = 0.009).ConclusionBCAAs are independently correlated with increased cIMT. This correlation would open a new field of research in the mechanistic understanding and risk assessment of CAD.

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography-tandem mass spectrometry.

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Automated isotope dilution liquid chromatography–tandem mass spectrometry with on-line dilution and solid phase extraction for the measurement of cortisol in human serum sample

A candidate reference measurement procedure involving automated isotope dilution coupled with liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS) with on-line dilution and solid phase extraction (SPE) has been developed and critically evaluated. We constructed the LC–MS/MS with on-line dilution and SPE system. An isotopically labelled internal standard, cortisol-d4, was added to serum sample. After equilibration, the methanol was added to the sample, and deproteination was performed. Then, the sample was applied to the LC–MS/MS system. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 1ngg−1, respectively. Excellent precision was obtained with within-day variation (RSD) of 1.9% for ID-LC–MS/MS analysis (n=6). This method, which demonstrates simple, easy, good accuracy, high precision, and is free from interferences from structural analogues, qualifies as a reference measurement procedure.