Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A™ column with mobile phase consisting of 0.05 M citric acid–0.1 M dipotassium hydrogen phosphate (pH 4.0)–methanol (97+3). The detection limits were 100–1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2–22.2 pmol/ml), ADP (5.5–22.2 pmol/ml), AMP (0.8–3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

A new selective pre-column ninhydrin-based derivatization for a RP-HPLC determination of plasma asymmetric dimethyl-l-arginine (ADMA) by fluorescence detection

We report a new selective and direct pre-column ninhydrin-based derivatization reaction for determination of plasma ADMA levels. This original derivatization procedure matched to a validated and rapid RP-HPLC method can be a useful alternative to other assays in which time consuming and expensive extraction and/or purification steps are required.     

Utility of green chemistry for sustainable fluorescence derivatization approach for spectrofluorimetric quantification of Darolutamide as antineoplastic drug in pharmaceutical formulation and spiked human plasma

Darolutamide is an oral nonsteroidal androgen receptor antagonist used to delay the process of prostate cancer to metastatic disease and to increase the quality of life for people with advanced prostate cancer. Here, a second spectrofluorimetric method was advanced for quantifying Darolutamide in pharmaceutical formulation and spiked human plasma. This method depends on the fluorescence derivatization of Darolutamide with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) at 75°C in a (pH 9) of borate buffer to produce a fluorescent derivative that can be detected at 520 nm after excitation at 460 nm. The method has been validated using ICH criteria, and it demonstrated linearity in the range 5–200 ng ml⁻¹_. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.15 and 3.84 nm, respectively. The proposed method was applied precisely and accurately for quantifying Darolutamide within the pharmaceutical formulation and spiking human plasma without any interferences. Moreover, the method's sustainability was evaluated and compared with the published method using two greenness assessment tools termed analytical eco-scale and Analytical GREEnness (AGREE). These findings suggest that the method is more sustainable than the published method.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

Determination of trace amino acids in human serum by a selective and sensitive pre-column derivatization method using HPLC-FLD-MS/MS and derivatization optimization by response surface methodology

Analysis of trace amino acids (AA) in physiological fluids has received more attention, because the analysis of these compounds could provide fundamental and important information for medical, biological, and clinical researches. More accurate method for the determination of those compounds is highly desirable and valuable. In the present study, we developed a selective and sensitive method for trace AA determination in biological samples using 2-[2-(7H-dibenzo [a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC) as labeling reagent by HPLC-FLD-MS/MS. Response surface methodology (RSM) was first employed to optimize the derivatization reaction between DBCEC and AA. Compared with traditional single-factor design, RSM was capable of lessening laborious, time and reagents consumption. The complete derivatization can be achieved within 6.3 min at room temperature. In conjunction with a gradient elution, a baseline resolution of 20 AA containing acidic, neutral, and basic AA was achieved on a reversed-phase Hypersil BDS C₁₈ column. This method showed excellent reproducibility and correlation coefficient, and offered the exciting detection limits of 0.19–1.17 fmol/μL. The developed method was successfully applied to determinate AA in human serum. The sensitive and prognostic index of serum AA for liver diseases has also been discussed.     

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicylic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde—3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.     

Determination of amino acids in human plasma by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination

Amino acids in human plasma were determined by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination. S-Carboxymethyl-l-cysteine, d-phenylglycine and S-aminoethyl-l-cysteine were found to be suitable internal standards. The proposed method is simple, rapid (deproteination time less than 1 min) and reproducible [relative standard deviation below 3% except for low-level aspartic acid (n = 3)]. The average recovery of 25 amino acids was above 90%. The elution time of amino acids in human plasma was approximately 2 h. Protein binding of tryptophan was also determined by the proposed method. The analytical data for amino acids in human plasma deproteinated using the proposed and published methods (5-sulphosalicylic acid and ethanol) were compared.     

High-sensitivity amino acid analysis by derivatization with O-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: applications in protein structure determination

An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.     

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.     

Engineered protein function by selective amino acid diversification

Almost all protein engineering methods rely upon making changes to naturally occurring proteins that already possess some of the desired properties. This will probably remain the case as long as we lack a complete understanding of the way that an amino acid sequence gives rise to a protein with a precisely defined biological function. Common to all methods for altering an existing protein is the selection of a subset of amino acids in the protein for variation and a choice of which substitutions to make at each position. Variants are then tested empirically and further variants are created based upon their performance. Differences between protein engineering methods are the ways in which amino acids are chosen for variation, the protocols followed for creating the variants, and how information regarding variant properties is used in creating subsequent variants. In this article, we describe these differences and provide examples of how the experimental parameters of specific projects determine which method is most suitable.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10–10 000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17°C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Determination of busulfan in human plasma using high-performance liquid chromatography with pre-column derivatization and fluorescence detection

A rapid, sensitive and reproducible high-performance liquid chromatographic assay for busulfan in human plasma was developed. After extraction of plasma samples with acetonitrile and methylene chloride, busulfan and the internal standard [1,5-bis(methanesulfonyloxy)pentane] were derivatized with 8-mercaptoquinoline to yield fluorescent compounds which were detected with a fluorescence detector equipped with filters of 360 nm (excitation) and 425 nm (emission). Calibration graphs showed a linear correlation (r>0.9990) over the concentration range of 20–2000 ng/ml. The recovery of busulfan from plasma standards was 70±5%. The detection and quantification limits for busulfan in plasma samples were established at 9 ng/ml and 20 ng/ml, respectively. The intra- and inter-assay variations were lower than 8% and 10%, respectively. The applicability of the method was verified by analyzing the plasma concentrations of busulfan in a patient to whom it was administered orally on two different days.     

Liquid chromatography method for determination of bivalirudin in human plasma and urine using automated ortho-phthalaldehyde derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method was developed using solid-phase extraction, o-phthalaldehyde (OPA) derivatization and fluorescence detection for the determination of the direct thrombin inhibitor bivalirudin in human plasma and urine. The use of this assay will facilitate the study of the pharmacodynamics of bivalirudin in studies of special patient populations. A C(18) bioanalytical column at a flow rate of 1 ml/min with an aqueous trifluoroacetic acid (0.1% TFA in deionized water, pH 2.2, v/v) mobile phase and methanol gradient was used. The assay demonstrated linearity from 3 to 20 microg/ml bivalirudin in plasma, with a detection limit of 1 microg/ml. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of bivalirudin in patients undergoing percutaneous coronary interventions (PCIs).     

Sensitive determination of trimetazidine in spiked human plasma by HPLC with fluorescence detection after pre-column derivatization with 9-fluorenylmethyl chloroformate

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 degrees C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm x 4.6 mm, i.d., 5 microm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5-200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5+/-1.3% (n=6; RSD=2.1%).     

实验性糖尿病小鼠的血清氨基酸代谢谱

目的通过测定2型糖尿病小鼠血清氨基酸代谢谱的变化,探讨代谢轮廓分析结合模式识别技术在糖尿病动物模型中的应用。方法 SPF级雄KM小鼠高脂饲料喂养4周后,腹腔注射链脲菌素(streptozotocin,STZ)建立2型糖尿病模型,动态监测空腹血糖(FBG)变化,分别于造模后第4周处死,收集小鼠血清,检测甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)水平。采用高效液相色谱(HPLC)柱前衍生方法检测小鼠血清中氨基酸代谢谱的变化。结果 2型糖尿病小鼠FBG、TG、TC明显升高,差异均有显著性。利用代谢轮廓分析可以对模型组大鼠代谢谱与对照组完全区分。结论小鼠成模后体内氨基酸发生了明显变化。从差异变量中鉴定出4个氨基酸对组间贡献较大(精氨酸、苯丙氨酸、赖氨酸、牛磺酸)。氨基酸的代谢轮廓分析结合模式识别技术可以在一定程度上反映2型糖尿病小鼠的代谢变化。     

Evidence for selective evolution in codon usage in conserved amino acid segments of human alphaherpesvirus proteins

Summary The genomes of human viruses herpes simplex 1 (HSV1) and varicella zoster (VZV), although similar in biology, largely concordant in gene order, and identical in many amino acid segments, differ widely in their genomic G+C (abbreviated S) content, which is high in HSV1 (68%) and low in VZV (46%). This paper analyzes several striking codon usage contrasts. The S difference in coding regions is dramatically large in codon site 3, S3, about 42%. The large difference in S3 is maintained at the same level in a subset of closely similar genes and even in corresponding identical amino acid blocks. A similar difference in S levels in silent site 1 (S1) is found in leucine and arginine. The difference in S3 levels occurs in every gene and in every multicodon amino acid form. The S difference also exists in amino acid usage, with HSV1 using significantly more codon types SSN, while VZV uses more codon types WWN (where W stands for A or T). The nonoverlapping and narrow histograms of S3 gene frequencies in both viruses suggest that the difference has arisen and been maintained by a process of selective rather than nonselective effects. This is in sharp contrast to the relatively large variance seen for highly similar genes in the human versus yeast analysis. Interpretations and hypotheses to explain the HSV1 vs VZV condon usage disparity relate to virus-host interactions, to the role of viral genes in DNA metabolism, to availability of molecular resources (molecular Gause exclusion principle), and to differences in genomic structure.     

Free amino acid quantification by LC-MS/MS using derivatization generated isotope-labelled standards

The further development of derivatizing reagents for plasma amino acid quantification by tandem mass spectrometry is described. The succinimide ester of 4-methylpiperazineacetic acid (MPAS), the iTRAQ reagent, was systematically modified to improve tandem mass spectrometer (MS/MS) product ion intensity. 4-Methylpiperazinebutyryl succinimide (MPBS) and dimethylaminobutyryl succinimide (DMABS) afforded one to two orders of magnitude greater MS/MS product ion signal intensity than the MPAS derivative for simple amino acids. CD(3) analogues of the modified derivatizing reagents were evaluated for preparation of amino acid isotope-labelled quantifying standards. Acceptable accuracy and precision was obtained with d(3)-DMABS as the amino acid standards derivatizing reagent. The product ion spectra of the DMABS amino acid derivatives are diagnostic for structural isomers including valine/norvaline, alanine/sarcosine and leucine/isoleucine. Improved analytical sensitivity and specificity afforded by these derivatives may help to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization generated isotope-labelled standards a viable alternative to amino acids analysers.     

Unprecedented derivatization of ferulic acid through selective methoxylation by Aspergillus brasiliensis ATCC 16404

Ferulic acid (FA) is a natural hydroxycinnamic acid widely found in medicinal and edible plants. Several experts have reported the biological potential of FA, including antioxidant and antimicrobial activities. The use of microorganisms in the derivatization of natural products is a useful and advantageous approach to the achievement of high value-added compounds. In order to access chemical derivatives, we conducted the biotransformation of FA by Aspergillus brasiliensis ATCC 16404 for 5 d. In the second day of fermentation, the FA was converted into the new (E)-3-(4-hydroxy-3-methoxyphenyl)-2-methoxyacrylic acid. This is the first time that the extended π-conjugation remained in the chemical structure after the biotransformation of FA. The cytotoxicities of FA and its derivative were evaluated. The biotransformation yielded a derivative less toxic than the parent compound.