Determination of 1-methylhistidine and 3-methylhistidine by capillary and chip electrophoresis with contactless conductivity detection

CE with capacitively coupled contactless detection (C4D) was used to determine 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH). The C4D response to 3-MH was studied in a BGE consisting of 500 mM acetic acid and ammonia at varying concentration and the results were compared with the theory. Complete separation of a model mixture of 3-MH, 1-MH, and histidine (His) was attained in two optimized BGEs, one containing 500 mM HAc, 20 mM NH4OH, and 0.1 % m/v hydroxyethylcellulose (HEC), pH 3.4 (I) and the other consisting of 100 mM morpholinoethanesulfonic acid (MES), 25 mM LiOH, and 0.1 % m/v HEC, pH 5.5 (II). These optimized BGEs were tested in CE/C4D analyses of urine. Promising results were obtained for separation and determination of 3-MH, 1-MH, and His on a silicon microchip, using aluminum strips as the C4D electrodes; the three analytes were baseline-separated within less than 30 s with a separation channel effective length of 38 mm. The LOD were satisfactory and amounted to 26.4 microM for 3-MH and 18.3 microM for 1-MH.     

Determination of 3-methylhistidine in hydrolysed proteins by fluorescamine derivatization and high-performance liquid chromatography

A method is described for the determination of the 3-methylhistidine content in myofibrilar proteins (myosin and actin) using reversed-phase high-performance liquid chromatography with ultraviolet detection. Proteins were hydrolysed and free amino acids were derivatized with fluorescamine. Elution was performed isocratically with acetonitrile-water (24:76). This method allows the detection of picomole amounts of 3-methylhistidine in myosin and actin.     

Quantification of histidine, 1-methylhistidine and 3-methylhistidine in plasma and urine by capillary electrophoresis UV-detection

We describe a new CE method with UV-detection for the quantification of histidine (His) and its methylated forms 1-methylhistidine and 3-methylhistidine, both in plasma and urine. Analytes were basically resolved using a 60?mmol/L Tris-phosphate run buffer pH 2.2 in less than 12?min. The use of a mixture of ACN/ammonia (80:20) for protein precipitation allows the quantitative recovery of all His from plasma. The optimization of the sample volume injection permits to reach an LOD of 20?nmol/L, thus improving the sensitivity of about hundred times in comparison to the previous described assays. Moreover, the opportunity to also measure creatinine in the same run makes it possible to evaluate the renal function contemporarily, thus avoiding further dosages with significant time saving. The application method has been proved by measuring His, 1-methylhistidine and 3-methylhistidine in 44 healthy subjects. In conclusion, our new method seems to be an inexpensive, fast and specific tool to assess large numbers of patients for routine analysis both in clinical and research laboratories.     

Isotachophoretic determination of 3-methylhistidine.

A capillary isotachophoretic (cITP) method to determine the concentration of 3-methylhistidine (3-MeHis) in meat and meat products is described. A clear separation of the 3-MeHis from histidine, 1-methylhistidine and other components of acidic sample hydrolyzate was achieved within 20 min. Method characteristics (linearity, accuracy, precision and detection limit) were determined. Low laboriousness, sufficient sensitivity and low running cost are the important attributes of cITP method. The developed method was successfully applied to analyses of real samples and used for the determination of lean meat content in meat and meat products.     

Rapid ion-exchange method for the determination of 3-methylhistidine in rat urine and skeletal muscle

A method for the determination of 3-methylhistidine using an automatic amino acid analyser has been developed. A single column system with lithium buffer (pH 3.950, 0.500 mol/l lithium and 0.067 mol/l citrate) was used for elution. The standard amino acid mixture of basic amino acids and some dipeptides usually present in physiological fluids was analysed for the development of the method. 3-Methylhistidine eluted in 46.7 +/- 0.049 min and the peak area coefficient of variation for the same sample was 1.07%. As 3-methylhistidine is completely resolved from the other basic amino acids and some dipeptides (anserine and carnosine), this method is suitable for the analysis of urine and muscle extracts as well as skeletal muscle protein hydrolysates where this amino acid is present in much lower concentrations than other amino acids.     

Some observations on the analytical utility of a fluorometric stopped-flow method for resolution of histidine and 1-methylhistidine mixtures

The fluorescent reactions of histidine and 1-methylhistidine with o-phthaldialdehyde and 2-mercaptoethanol are exploited for the individual and simultaneous determination of these compounds by the stopped-flow technique. The instrumental setup used consists of a stopped-flow module coupled to a conventional fluorescence spectrophotometer. The parameters used for these determinations are the initial rate and the fluorescence amplitude of the kinetic curves. Linear ranges, precision, and the results of the selectivity studies are reported. An extension of the proportional-equations method is used to resolve histidine: 1-methylhistidine mixtures in ratios between 3:1 and 1:8. The method is very simple, inexpensive, and fast (only about 10 sec is required for initial-rate measurements, and about 90 sec for those of signal amplitude).     

A sensitive and specific high performance liquid chromatographic assay for imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine.

A sensitive and specific high performance liquid chromatographic method is described for measuring imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine. Muscle extract, serum or urine was reacted with o-phthaldialdehyde and the derivatives were separated by reversed phase chromatography with column switching and fluorescence detection.     

Microemulsion electrokinetic chromatography: an application for the simultaneous determination of suspected fragrance allergens in rinse-off products

The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4-24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.     

Low-capacity cation-exchange chromatography of ultraviolet-absorbing urinary basic metabolites using a reversed-phase column coated with hexadecylsulfonate

A low-capacity cation-exchange HPLC method for the determination of UV-absorbing organic cations such as amino acids, histidine dipeptides, and creatinine was developed. A commercially available reversed-phase column was dynamically coated with hexadecylsulfonate, and was successfully used for the cation-exchange separation with ethylenediammonium eluting ion at pH 2.5. The coated column was enough stable for the specific use with a completely aqueous mobile phase at low and constant pH; and the day-to-day reproducibility for retention time was 0.9-1.7% of RSD (relative standard deviation). The linear relation between concentrations and detector responses (area) by using a photodiode-array UV detection at 210 nm ranged from 0.2 to 1000 microM (sample size 50 microl) for 1-methylhistidine, 3-methylhistidine, histidine, creatinine, anserine, carnosine, and homocarnosine, and from 0.5 to 2000 microM for creatine, tyrosine, and phenylalanine, with less than 5% of RSD. The UV spectrum (190-300 nm) obtained during chromatography was very indicative for each analyte. Overall recoveries were 97-104%. The developed HPLC method in conjunction with preliminary fractionation technique could be applied to the analysis of urine of patient with metabolic disorder such as phenylketonuria.     

Improved High Performance Liquid Chromatographic Method for the Quantification of 3-Methylhistidine in Serum and Urine

Abstract

We optimized an high-performance liquid chromatographic (HPLC) method for the determination of 3-methylhistidine (3-MeHis) in biological fluids. After pre-column derivatization with OPA, analytical separation was achieved on a reversed-phase C18 column by a simple gradient between sodium propionate buffer and acetonitrile. the method is accurate, reproducible and sensitive, and allows the determination of urinary 3-MeHis levels in about 55 min. Other additional 16 amino acids may be easily quantified while the 3-MeHis peak is well resolved from an unknown urinary compound potentially interfering.     

A simple gas chromatographic-mass spectrometric method for the concurrent determination of novel pentafluoropropionated derivatives of histidine,histamine and their 1-methyl metabolites

Summary Histidine, 1,4-methylhistidine, histamine and 1,4-methyl-histamine were reacted directly for 1.5 hours at 70°C with pentafluoropropionic anhydride (PFPA). No previous esterification of the carboxyl group is needed. The reaction mixture, taken to dryness and redissolved in acetonitrile, can be separated on 3% OV-17 (retention indices: 1736, 1582, 1827 and 1763) and 3% OV-225 (retention indices: 2355, 2015, 2443 and 2147). The derivatives thus obtained are stable for one week at –4°C. The mass spectra of pentafluoropropionated histidine and 1,4-methylhistidine show relatively abundant molecular ions at m/e 403 and m/e 417 respectively, which confirm the incorporation of two PFP groups in both cases. However, both of these m/e values are 44 mass units less than the calculated relative molecular masses, a fact that can be accounted for by an intramolecular decarboxylation during derivatization. Histamine and 1,4-methylhistamine give molecular ions at m/e 257 and m/e 271, corresponding to the calculated values of the monoacylated derivatives.     

Analysis of novel imidazoles from isolated perfused rabbit heart by two high-performance liquid chromatographic methods.

The paper reports two analytical high-performance liquid chromatographic methods to detect and quantify cardiac-derived histidyl derivatives. Method A relies on relative hydrophobicities as a basis of separation. Method B is an ion-pairing method in which the compounds are eluted in an entirely different order. Fractions collected from method A have been co-eluted in admixture in method B with authentic reference compounds. Thus the existence of the following imidazole ring-containing compounds derived from heart have been confirmed: N-acetylhistidine, N-acetyl-1-methylhistidine, N-acetylcarnosine, N-acetylhomocarnosine, homocarnosine, anserine, carnosine, balenine. Compounds were found in both tissue samples and perfusates.     

Analysis of 1- and 3-methylhistidines, aromatic and basic amino acids in rat and human urine

A procedure based on automated amino acid analysis has been developed to simultaneously quantify 1-methylhistidine (1-MH), 3-methylhistidine (3-MH), tyrosine, phenylalanine, tryptophan, lysine, histidine and arginine levels in human and rat urines. Deproteinized urine samples containing amino acids in the range 1-10 nmol were analyzed using single-column methodology with ninhydrin detection. Standard curves produced correlation coefficients greater than or equal to 0.99 with duplicate analyses agreeing to within +/- 1.9%. Quantitative recovery was ensured by using L-alpha-amino-beta-guanidinopropionic acid as an internal standard. Elution was accomplished in less than 90 min at pH 5.7 with sodium citrate buffers at 45 degrees C and 65 degrees C. Since 3-MH in the rat is acetylated at the alpha-amino group, rat, but not human, urine ultrafiltrates required acid hydrolysis prior to analysis. The utility of the technique of analysis of 1-MH and 3-MH in human urine was demonstrated for an adult male on a meat-free diet for 21 days; urinary excretion rates for 3-MH and 1-MH were determined to be 3.06 +/- 0.10 and 0.72 +/- 0.07 mumol/kg body mass/day, respectively. The technique was also used to measure the effect of disuse atrophy of rat skeletal muscle which induced a 40-60% increase in 3-MH. The procedure is also highly suited for measurement of urinary aromatic and/or basic amino acids.     

Pyrolysis—gas chromatography of histidine and 3-methylhistidine

Pyrolysis of histidine and 3-methylhistidine using a Curie-point analyser at 770°C yielded imidazole and 1-methylimidazole, respectively, which were identified by gas—liquid chromatography. The posibility of applying this reaction to the differentiation of vegetable and meat protein was examined.     

高效液相色谱法测定土壤中香豆素类灭鼠药残留

建立了测定土壤中杀鼠灵、杀鼠醚、溴敌隆、氟鼠灵、溴鼠隆5种香豆素类灭鼠药残留量的柱后衍生荧光检测高效液相色谱方法。样品在加入内标物氯杀鼠灵后用丙酮-氨水-甲醇（体积比为100：3：100）混合液提取,浓缩的提取液用5 mL正己烷-氯仿（体积比为3：1）混合液溶解,NH₂固相萃取小柱净化,用15 mL 50 mmol/L四丁基磷酸二氢铵甲醇溶液洗脱分析物,移除溶剂,用甲醇-0.25%（体积分数）乙酸水溶液（体积比为3：2）混合液溶解,过滤后,经高效液相色谱分离,以甲醇-氨水-水（体积比为1：1：8）混合液为衍生试剂进行柱后衍生,采用荧光检测器检测。杀鼠灵、杀鼠醚、溴敌隆、氟鼠灵、溴鼠隆5种鼠药在0.02～10.00 mg/L范围内线性关系良好,相关系数均大于0.999,定量限（LOQ,S/N=10）为2.2～18.5 μg/L。在0.1～0.3 mg/kg添加水平内,5种灭鼠药的回收率为94.6%～118.0%,相对标准偏差（RSD）为0.8%～10.2% （n=3）。实验结果表明该方法灵敏、准确,重复性好。     

高效液相色谱-紫外法快速测定塑料类食品接触材料及制品中7种对苯二甲酸酯或苯甲酸酯的特定迁移量

建立了高效液相色谱-紫外检测（HPLC-UV）法快速测定从塑料类食品接触材料及制品迁移至10%（v/v）乙醇、3%（m/v,即3 g/100 mL）乙酸、4%（v/v）乙酸、20%（v/v）乙醇、50%（v/v）乙醇、95%（v/v）乙醇和橄榄油7种食品模拟物中对苯二甲酸二甲酯、对苯二甲酸二辛酯、苯甲酸甲酯、苯甲酸乙酯、苯甲酸丙酯、苯甲酸丁酯和新戊二醇二苯甲酸酯的特定迁移量。考察了多种提取溶剂、QuEChERS dSPE EMR-Lipid试剂盒和Captiva EMR-Lipid试剂盒对橄榄油食品模拟物中7种对苯二甲酸酯或苯甲酸酯的提取或净化效果。以甲醇和水为流动相进行梯度洗脱,7种对苯二甲酸酯或苯甲酸酯在苯基柱上于17 min内达到基线分离。检测波长为237 nm,进样量为10 μL。7种对苯二甲酸酯或苯甲酸酯在7种食品模拟物中的定量限为0.2~8.1 mg/kg、1~80 mg/L或8~160 mg/kg,相关系数r≥0.9998。在2或8、60、80或160 mg/kg 3个加标水平的回收率为91.7%~106%,相对标准偏差为0.1%~3.1%。该方法样品前处理简便,色谱分离和线性关系好,回收率和重复性较好,已应用于实际样品的检测。     

液相色谱-电喷雾串联质谱法测定生姜中的215种农药残留

建立了生姜中215种农药多残留测定的液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)方法。样品用1%醋酸-乙腈溶液均质提取,经Sep-Pak Vac固相萃取柱净化,乙腈-甲苯(3:1, v/v)洗脱,旋转蒸发浓缩至约0.5 mL后,于室温氮气吹干,用乙腈-水(3:2, v/v)溶解,以电喷雾电离串联质谱在正离子多反应监测(MRM)模式下进行测定。在定量限水平进行添加回收率实验,方法的回收率范围为68.1%～132.6%,其中回收率在70%～120%的占94.4%,相对标准偏差(RSD)范围为0.4%～25.0%。方法的检出限(S/N=3)和定量限(S/N=10)范围分别为0.01～70.45 μg/L和0.04～234.84 μg/L。该方法操作简便,灵敏度、准确度和精密度均符合农药多残留检测技术要求,适用于生姜中215种农药多残留的快速测定。     

The NaK 1 1,3delta states: theoretical and experimental studies of fine and hyperfine structure of rovibrational levels near the dissociation limit

Earlier high-resolution spectroscopic studies of the fine and hyperfine structure of rovibrational levels of the 1 3delta state of NaK have been extended to include high lying rovibrational levels with v < or = 59, of which the highest levels lie within approximately 4 cm(-1) of the dissociation limit. A potential curve is determined using the inverted perturbation approximation method that reproduces these levels to an accuracy of approximately 0.026 cm(-1). For the largest values of v, the outer turning points occur near R approximately 12.7 angstroms, which is sufficiently large to permit the estimation of the C6 coefficient for this state. The fine and hyperfine structure of the 1 3delta rovibrational levels has been fit using the matrix diagonalization method that has been applied to other states of NaK, leading to values of the spin-orbit coupling constant A(v) and the Fermi contact constant b(F). New values determined for v < or = 33 are consistent with values determined by a simpler method and reported earlier. The measured fine and hyperfine structure for v in the range 44 < or = v < or = 49 exhibits anomalous behavior whose origin is believed to be the mixing between the 1 3delta and 1 1delta states. The matrix diagonalization method has been extended to treat this interaction, and the results provide an accurate representation of the complicated patterns that arise. The analysis leads to accurate values for A(v) and b(F) for all values of v < or = 49. For higher v (50 < or = v < or = 59), several rovibrational levels have been assigned, but the pattern of fine and hyperfine structure is difficult to interpret. Some of the observed features may arise from effects not included in the current model.     

大孔树脂-高速逆流色谱分离纯化薇甘菊中的黄酮类化合物

该文建立了大孔树脂-高速逆流色谱分离薇甘菊中黄酮类物质的方法。分离条件为:采用大孔树脂AB-8,洗脱液为50%（v/v）乙醇水溶液,高速逆流色谱溶剂体系为正丁醇-乙酸-水（4：1：5,v/v）。从薇甘菊中分离到4种黄酮类物质:槲皮素-3-O-芸香糖苷（纯度90.2%）、山奈酚-3-O-芸香糖苷（纯度98.55%）、木犀草苷（纯度98.33%）和紫云英苷（纯度99.23%）。建立的大孔树脂-高速逆流色谱方法简单、高效,可扩展应用于从其他植物中分离黄酮类物质。     

High-performance liquid chromatographic determination of acetone in blood and urine in the clinical diagnostic laboratory.

A method for the determination of acetone in plasma or urine by high-performance liquid chromatography (HPLC) was developed. Plasma specimens are deproteinized with acetonitrile (1:1, v/v) 2,4-dinitrophenylhydrazine (DNPH) is added to the supernatant or to filtered urine samples, similarly treated with acetonitrile (2:1, v/v) to prevent crystallization of the synthesized phenylhydrazone. An aliquot (20 microliters) of the reaction mixture was subjected to HPLC at ambient temperature using a reversed-phase Pecosphere 3 x 3 C18 column with acetonitrile-water (45:55, v/v) as eluent at a flow-rate of 1 ml/min and detection at 365 nm. Hydroxyacetone and acetoacetate phenylhydrazone derivatives do not interfere. The identification of acetone by its retention time was confirmed by comparison with a laboratory-synthesized acetone DNPH derivative. The concentration of acetone, eluted within 3 min, was determined by the peak-height method. The detection limit was 0.034 mmol/l; the relative standard deviations were less than 5% within run (n = 20) and less than 10% between run (n = 20).