Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography–tandem mass spectrometry

Since sarcosine and d,l-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine–alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R ²?=?0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of d,l-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb–1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from d,l-alanine leading to underivatized quantification of sarcosine.

肌氨酸的二硫代甲酰化及其电位滴定分析

报道了二硫化碳对肌氨酸（ＳＡ）中仲胺基的二硫代甲酰化（ＤＴＦ）反应及其在ＳＡ定量分析中的应用。上述反应中生成的二硫代甲酰胺（ＤＴＦＡ）基团可以通过电位滴定确定含量，从而求得ＳＡ含量。加入回收试验表明该法对ＳＡ的适宜检测浓度范围为１￣１５ｇ／Ｌ；相对标准偏差〈１．０％。该法用于化学合成ＳＡ的含量测定，简便、快捷，准确。     

Preparation and Crystal Structure of Sarcosine 5-Nitrosalicylic Acid Organic Adduct

1 INTRODUCTION Nonlinear optical second harmonic generation(SHG) material is one important embranchment ofnonlinear optical materials. The main advantages oforganic nonlinear optical materials over the inor-ganic ones are as follows: bigger nonline…     

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H₂O₂ (at 0.6 V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16 nM, using a linear concentration range between 10 and 100 nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.     

Colorimetric determination of sarcosine in urine samples of prostatic carcinoma by mimic enzyme palladium nanoparticles

The proposed palladium nanoparticles (Pd NPs), which with the catalytic activity similar to the horseradish peroxidase (HRP) mimic enzyme, can effectively catalyze the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine sulfate (TMB) accompanied with a color change from colorless to blue in solution. And as a result, the sensitive detection of sarcosine can be realized by the naked eye observation and ultraviolet spectrophotometry, using Pd NPs as catalyst and TMB as the substrate of the simulation enzyme catalytic reaction. Under the optimal condition, the catalytic system of Pd NPs mimic enzyme can be used for the detection of sarcosine. It has been found that the color change could be clearly observed with the naked eyes, and the absorbance intensity at 653 nm showed a fine linear fitting with the concentration of sarcosine in the range from 0.01 μM to 50 μM, and the detection limit (3σ/S) for sarcosine was calculated to be 5.0 nM. In order to evaluate the feasibility and reliability, the method was also used for analyzing concentrations of sarcosine in human urine samples from diagnosed prostate cancer patients and healthy donors. It is expected to provide a convenient and efficient method for indirect evaluation for the diagnosis of prostatic carcinoma (PCa).     

Sensitive determination of the potential biomarker sarcosine for prostate cancer by LC–MS with N,N′‐dicyclohexylcarbodiimide derivatization

Sarcosine, a potential biomarker of prostate cancer, has drawn great attention in recent years. However, controversial research keeps arising about its role as a biomarker that might come from the two isomers (α‐alanine and β‐alanine) of sarcosine due to their same molecular weight and similar properties, which could interfere with the accurate detection of sarcosine. In this study, a simple and sensitive method was developed for the detection of sarcosine and the two isomers by LC with ion‐trap MS through a novel derivatization reagent N,N′‐dicyclohexylcarbodiimide. N,N′‐Dicyclohexylcarbodiimide is usually considered as a condensation reagent, however, it was directly used as a derivatization reagent through a rearrangement side reaction in this study. The proposed method not only improved the chromatographic retention behavior of sarcosine and its two isomers, which was a benefit to their separation, but also dramatically enhanced the detection sensitivity of sarcosine, which was more favorable for real sample analysis. The factors affecting the productivity of the derivatization reaction, such as reaction time and amount of derivatization reagent, were systematically optimized. The method shows good linearity (R² > 0.99), sensitivity with LODs of sarcosine as low as 1 ng/mL, and repeatability with the RSD < 6.07%. The developed method was applied to the analysis of urine.     

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5?×?10(-7)?M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.     

Density,viscosity, and N2O solubility of aqueous amino acid salt and amine amino acid salt solutions

Physicochemical properties of aqueous amino acid salt (AAS), potassium salt of sarcosine (KSAR) and aqueous amine amino acid salt (AAAS), 3-(methylamino)propylamine/sarcosine (SARMAPA) have been studied. Densities of KSAR were measured for sarcosine mole fraction 0.02 to 0.25 for temperature range 298.15 K to 353.15 K, the viscosities were measured for 0.02 to 0.10 mole fraction sarcosine (293.15 K to 343.15 K) while the N₂O solubilities were measured from 0.02 to 0.10 mole fraction sarcosine solutions (298.15 K to 363.15 K). Densities of SARMAPA were measured for sarcosine mole fraction 0.02 to 0.23 for temperature range (298.15 K to 353.15 K), viscosities were measured for 0.02 to 0.16 mole fraction sarcosine (293.15 K to 343.15 K) while the N₂O solubilities were measured from 0.02 to 0.16 mole fraction sarcosine solutions (298.15 K to 343.15 K). Experimental results were correlated well with empirical correlations and N₂O solubility results for KSAR were predicted adequately by a Schumpe model. The solubilities of N₂O in AAS and AAAS are significantly lower than values for amines. The solubilities vary as: amine > AAAS > AAS.     

A Novel Derivatization Method for Separation of Sarcosine from Isobaric l-Alanine in Human Urine by GC–MS

Sarcosine, an isomer of l-alanine, has been recently proposed as a potential biomarker for prostate cancer risk and aggressiveness, while some studies debated its importance. As both sarcosine and l-alanine are present in human urine, it is a great challenge to separate and accurately quantify these isobaric (i.e., same m/z) compounds by chromatographic separation and mass spectrometric detection. In this study, we developed a novel 1,3-dipolar cycloaddition derivatization method that resolves sarcosine from l-alanine and allows accurate quantification of sarcosine in human urine by gas chromatography–mass spectrometry (GC–MS). This novel derivatization approach was specific to sarcosine only, while the common silylanization method resulted in overlapped derivates of both sarcosine and l-alanine. The derivatization conditions, including reagent amount, reaction temperature and time, were optimized. The method developed here has excellent precision (relative standard deviation <4.7 %, n = 5), good linearity (slope = 0.2408; r ² = 0.9996, 0.1–100 μg mL^?1), and a low limit of detection in human urine (0.15 ng mL^?1). Application of this analytical method to urine samples spiked with standard sarcosine indicates that it is a robust and powerful alternative for resolving and quantifying sarcosine from l-alanine isomer in human urine by GC–MS.     

Nonderivatized Sarcosine Analysis by Gas Chromatography after Solid-Phase Microextraction by Newly Synthesized Monolithic Molecularly Imprinted Polymer

In this study, a simple method for analysis of nonderivatized sarcosine was developed by gas chromatography. It is based on solid-phase microextraction of sarcosine on a novel synthesized solid-phase microextraction (SPME) fiber. A monolithic SPME fiber was fabricated based on a molecularly imprinted polymer that could be coupled with gas chromatography for extraction and determination of sarcosine. Extraction time, pH, and ionic strength were investigated as important factors in the extraction procedure. The fabricated fiber was firm, inexpensive, stable, and selective, which are vital characteristics for SPME. The selectivity of the fabricated fiber in relation to analog compounds was also investigated. Under optimum conditions, the calibration curve was linear in the range of 1–100 mg L⁻¹ (R ² = 0.987). High extraction efficiency for sarcosine was obtained with a detection limit of 0.37 mg L⁻¹. The fabricated fiber was successfully applied for SPME of sarcosine from urine after its extraction, followed by gas chromatography flame ionization detector analysis.

     

Hydrogen‐deuterium exchange of α‐carbon protons and fragmentation pathways in N‐methylated glycine and alanine‐containing peptides derivatized by quaternary ammonium salts

Recently, we developed a selective and efficient method of hydrogen‐deuterium exchange (HDX) at the α‐carbon (α‐C) of sarcosine residue (N‐methylglycine) in model peptides [B?chor et al. J. Mass Spectrom. 2014, 49, 43]. Here, we report the influence of quaternary ammonium (QA) group on HDX at the α‐C of sarcosine and N‐methylalanine in peptides. The obtained results suggest a significant acceleration of the HDX in sarcosine residue caused by the presence of QA. The effect depends on the distance between the sarcosine residue and QA moiety. The deuterons, introduced at α‐C, are resistant to the back‐exchange in acidic aqueous solution. The collision induced dissociation of the deuterium‐labeled analogs of QA‐tagged oligosarcosine peptides without mobile hydrogen revealed the mobilization of the hydrogens localized at α‐C of sarcosine residue. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular recognition in the gas phase ligand switching reactions of the proton bound dimer of sarcosine and glycylglycine

Ion-molecule reactions of the proton bound dimer of [Sar+H+GlyGly]+ (where Sar = sarcosine and GlyGly = glycylglycine) proceed via two main reaction channels, i.e. association and ligand switching. The association reaction, which involves formation of an adduct between the protonated dimer and neutral base, occurs more readily for the oxygen containing bases and those with a lower gas phase basicity. Molecular recognition was demonstrated for the ligand switching reactions in which nitrogen containing bases preferred to switch out sarcosine while the oxygen containing bases preferred to switch out glycylglycine. Molecular dynamics followed by semiempirical PM3 calculations for the ligand switching reactions of [Sar+H+GlyGly]+ with methylamine directly correlated with the experimental findings by predicting that the most stable product ion arises from switching out sarcosine. These calculations reveal that the most stable adduct structure and the most stable ligand switched structure arise from proton transfer to methylamine to yield ions of the type [(Sar)(GlyGly)(LigH+)] and [(GlyGly)(LigH+)].     

Fully automated solid-phase microextraction-fast gas chromatography-mass spectrometry method using a new ionic liquid column for high-throughput analysis of sarcosine and N-ethylglycine in human urine and urinary sediments

A fully automated, non invasive, rapid and high-throughput method for the direct determination of sarcosine and N-ethylglycine in urine and urinary sediments using hexyl chloroformate derivatization followed by direct immersion solid-phase micro extraction and fast gas chromatography-mass spectrometric analysis was developed and validated. The use of a new ionic liquid narrow bore column, as well as the automation and miniaturization of the preparation procedure by a customized configuration of the utilized XYZ robotic system, allowed a friendly use of the GC apparatus achieving a quantitation limit of 0.06 μg L(-1) for sarcosine, good repeatability with CV always lower than 7% and reduced analysis times useful for point-of-care testing. The method was then applied for the analysis of 56 samples of urine and urinary sediments in healthy subjects, in those with benign prostatic hypertrophy and in patients with clinically localized prostate cancer. The results obtained showed that the medians of sarcosine/creatinine in urine were 103, 137 and 267 μg g(-1) respectively, thus assessing the potential use of sarcosine as urinary biomarker for prostate cancer detection. The highest values of sensitivity (79%) and specificity (87%) were obtained in correspondence of a cut-off value of 179 μg sarcosine(g creatinine)(-1), thus by using this cut-off threshold, sarcosine was significantly associated with the presence of cancer (p<0.0001). Finally, ROC analyses proved that the discrimination between clinically localized prostate cancer and patients without evidence of tumor is significantly correlated with sarcosine.     

基于碳量子点过氧化物模拟酶的肌氨酸比色测定

以果皮为原料制备了水溶性碳量子点(CQDs), 透射电子显微镜分析表明其平均粒径为2.4 nm; 红外光谱分析证实CQDs表面富含含氧官能团. 所得CQDs可催化H₂O₂氧化3,3',5,5'-四甲基联苯胺(TMB)生成蓝色氧化产物(ox-TMB). 基于上述反应, 建立了测定H₂O₂的分析方法, 在pH=3.5, 温度40 ℃, TMB浓度为1.0 mmol/L及孵育时间15 min的条件下, 当H₂O₂浓度在5.0~100 μmol/L范围时, ox-TMB的吸光度随H₂O₂浓度的升高而线 性增强, 方法的检出限为3.0 μmol/L. 鉴于H₂O₂是肌氨酸氧化酶催化肌氨酸氧化的产物之一, 建立了CQDs催化H₂O₂氧化TMB间接测定肌氨酸的分析方法. 结果表明, 当肌氨酸浓度在0.5~350 μmol/L范围内时, 吸光度 与其浓度呈良好的线性关系, 方法的检出限为0.3 μmol/L; 将该方法应用于人体尿样的测定, 回收率为94%~99%.     

Kinetics and Mechanism of Oxidation of Sarcosine by Diperiodatocuprate(III) Complex in Alkaline Medium

The kinetics of oxidation of sarcosine by diperiodatocuprate(III) (DPC) was studied with spectrophotometry in a temperature range of 292.2–304.2 K. The reaction between diperiodatocuprate(III) and sarcosine in alkaline medium exhibits 1:1 stoichiometry (DPC:sarcosine). The reaction was found to be first order with respect to both DPC and sarcosine. The observed rate constant (k_obs) decreased with the increase of the [IO^?₄], decreased with the increase of the [OH^?], and then increased with the increase of the [OH^?] after a turning point. There was no salt effect, and free radicals were detected. Based on the experimental results, a mechanism involving the diperiodatocuprate(III) (DPC) as the reactive species of the oxidant has been proposed. The activation parameters, as well as the rate constants of the rate‐determining step, have been calculated.     

Amperometric Sarcosine Biosensors Based on Electrodeposited Conductive Films Contain Indole-6-carboxylic Acid

Sarcosine level in serum is of important clinical significance in distinguishing prostate cancer. This work depicts an amperometric sarcosine biosensor with good anti-interference performance by electro-codepositing manganese phosphate, 3,4-ethylenedioxythiophene (EDOT) and indole-6-carboxylic acid (IA) on the glass carbon electrode. The prepared sarcosine biosensor has a wide linear detection range (1–55 μM) with a low detection limit of 0.11 μM. This work provides an anti-interference approach by controlling the surface charge density of the biosensor to sarcosine sensing, which has great potential to be used as point of care testing (POCT) device for the rapid detection of prostate cancer biomarkers.     

GC/MS-based metabolomic approach to validate the role of urinary sarcosine and target biomarkers for human prostate cancer by microwave-assisted derivatization

A recent study showed that sarcosine may be potentially useful for the diagnosis and prognosis of prostate cancer (PCa). The aim of this study was to validate diagnostic value of sarcosine for PCa, to evaluate urine metabolomic profiles in patients with PCa in comparison of non-cancerous control, and to further explore the other potential metabolic biomarkers for PCa. Isotope dilution gas chromatography/mass spectrometry (ID GC/MS) metabolomic approach was applied to evaluate sarcosine using [methyl-D₃]-sarcosine as an internal standard. Microwave-assisted derivatization (MAD) together with GC/MS was utilized to obtain the urinary metabolomic information in 20 PCa patients compared with eight patients with benign prostate hypertrophy and 20 healthy men. Acquired metabolomic data were analyzed using a two-sample t test. Diagnostic models for PCa were constructed using principal component analysis and were assessed with receiver–operating characteristic curves. Results showed that the urinary sarcosine level has no statistical difference between the PCa group and the control group. In addition, nine metabolomic markers between the PCa group and the healthy male group were selected, which constructed a diagnostic model with a high area under the curve value of 0.9425. We conclude that although urinary sarcosine value has limited potential in the diagnostic algorithm of PCa, urinary metabolomic panel based on GC/MS assay following MAD may potentially become a diagnostic tool for PCa.     

Diaquaiodidotetrasarcosinepotassium: an overview of sarcosine metal halogenide structures

The monoclinic crystal structure of tetrasarcosine potassium iodide dihydrate {or catena‐poly[[potassium‐tetra‐μ‐sarcosine‐κ⁴O:O′;κ⁴O:O] iodide dihydrate]}, {[K(C₃H₇NO₂)₄]I·2H₂O}_n or Sar₄·KI·2H₂O (space group C2/c), comprises two crystallographically different sarcosine molecules and one water molecule on general positions, and one K⁺ cation and one I⁻ anion located on twofold axes. The irregular eight‐coordinated K⁺ polyhedra are connected into infinite chains along [001] via sarcosine molecules. This compound is the first sarcosine metal halogenide salt with a 4:1 ratio. A short overview of other sarcosine metal halogenide salts is presented and relationships to similar glycine salts are discussed.     

An improved synthesis of 1-methyl-2,5-piperazinedione

A new synthesis of 1-methyl-2,5-piperazinedione in three steps starting from sarcosine is described. This method proceeds in higher overall yield (49%) than previous methods.     

Single crystal EPR studies on Mn(II)-doped sarcosine cadmium chloride and sarcosine cadmium bromide: study of zero-field splitting tensor in iso-structural complexes

EPR spectra of single crystals of Mn(II)-doped sarcosine cadmium chloride and sarcosine cadmium bromide are studied in Q-band and in X-band at room temperature. Two magnetically inequivalent sites are observed in both the lattices in a distorted octahedral environment. The spin-Hamiltonian parameters are extracted and are found to have a rhombic symmetry. The angular variation of the zero-field transitions is simulated for one of the sites with an asymmetric zero-field tensor D = 480 x 10(-4) cm(-1), E = -115 x 10(-4) cm(-1) and a = 10 x 10(-4) cm(-1) for Mn(II) in sarcosine cadmium chloride and with D = 460 x 10(-4) cm(-1) E = -98 x 10(-4) cm(-1) and a = 10 x 10(-4) cm(-1) for Mn(II) in sarcosine cadmium bromide. The observed large value of zero-field tensor is due to the steric effects of the crystal packing caused by the ligands. Matumura's plot predicts an average covalency of 8.8 and 7.7% for the manganese-ligand bond in SCC and SCB lattices respectively.