Determination of ethyl glucuronide in hair samples of Chinese people by protein precipitation (PPT) and large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS)

Ethyl glucuronide (EtG) has been shown to be a suitable marker of excessive alcohol consumption. Determination of EtG in hair samples may help to differentiate social drinkers from alcoholics, and this testing can be widely used in forensic science, treatment programs, workplaces, military bases as well as driving ability test to provide legal proof of drinking. A method for determination of EtG in hair samples using large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS) was developed and validated. Hair samples (in 1 mL deionized water) were ultrasonicated for 1h and incubated overnight; these samples were then deproteinated to remove impurities and derivatisated with 15 μL of pyridine and 30 μL of BSTFA. EtG was detected using GC/MS/MS in multiple-reaction monitoring mode. This method exhibited good linearity: y=0.0036 x+0.0437, R2=0.9993, the limit of detection and the limit of quantification were 5 pg/mg and 10 pg/mg, respectively. The extraction recoveries were more than 60%, and the inter-day and intra-day relative standard deviations (RSD) were less than 15%. This method has been applied to the analysis of EtG in hair samples from 21 Chinese subjects. The results for samples obtained from all of those who were teetotallers were negative, and the results for the other 15 samples ranged from 10 to 78 pg/mg, except for one negative sample. These data are the basis for interpretation of alcohol abuse.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

Quantitative proteomic analysis of ribosomal protein L35b mutant of Saccharomyces cerevisiae

Recent studies have revealed that in higher eukaryotes, several ribosomal proteins are involved in some pathological events or developmental defects, indicating that ribosomal proteins perform unconventional functions other than protein biosynthesis. To obtain an insight into the novel roles of ribosomal proteins, we aimed to analyze the changes in proteome expression in ribosomal protein mutants by using Saccharomyces cerevisiae as a model system. We introduced the rpl35bΔ mutation into the 4159 green fluorescent protein (GFP)-tagged yeast strains by using the synthetic genetic array (SGA) method, and performed quantitative proteomic analysis by using a multilabel microplate reader and flow cytometer. We identified 22 upregulated and 20 downregulated proteins in the rpl35bΔ mutant. These proteins were primarily classified into the Gene Ontology (GO) categories of cellular biosynthetic process, translation, protein or nucleotide metabolic process, cell wall organization and biogenesis, and hyperosmotic response. We also investigated the correlation between the mRNA and protein levels of the identified proteins. Our results show that a ribosomal protein mutation can lead to perturbation in the expression of several proteins, including some other ribosomal proteins. Furthermore, our approach of combining a library of GFP-tagged yeast strains and the SGA method provides an effective and highly sensitive method for dynamic analysis of the effects of various mutations on proteome expression.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: Total and free testosterone reference ranges

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 μL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y = 1.02x + 1.5, r² = 0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.     

串联质谱图谱从头测序算法研究进展

近年来,基于质谱技术的高通量蛋白质组学研究发展迅速,利用串联质谱图谱鉴定蛋白质是其数据处理中一个基础而又重要的环节．由于不需要利用蛋白质序列数据库,从头测序方法能够分析新物种或者基因组未测序物种的串联质谱数据,具有数据库搜索方法不可替代的优势．简要介绍高通量串联质谱图谱从头测序问题及其研究现状．归纳出几种典型的计算策略并分析了各种策略的优缺点．总结常用的从头测序算法和软件,介绍算法评估的各种指标和常用评估数据集,概括各种算法的特点,展望未来研究可能的发展方向．