Amino Acids In Biological Fluids Research Articles

Diagnostic analysis of toxic damage due to a dose of zoledronic acid

Introduction: Comprehensive analysis is necessary to analyze the widespread use of bisphosphonate drugs in the treatment and prevention of various diseases of connective tissue structures. The relevance of the study depends on the impact they have on the patient’s somatic condition, given the number of early and late complications diagnosed by domestic and foreign clinicians.
 Materials and methods: The methodology used was developed on Stavropol Regional Clinical Hospital from 2010 to 2022. A randomized and observational study of the state of connective tissue structures was conducted in 186 patients who had a history of taking zoledronic acid and developed bisphosphonate osteonecrosis of the jawbone. At the diagnostic and treatment stages, patients were tested using laboratory methods to monitor amino acids in biological fluids (blood), as well as bone blocks extracted intraoperatively using highly effective gas-liquid chromatography on AAA 500.
 Results and discussion: Aminograms obtained in the pre-operative period indicate toxic effects of zoledronic acid on neuro, endocrine, and other human systems. The initiation and progression of aseptic inflammatory processes in tissue structures is correlated with the deteriorating general ocular status of patients when amino acids, such as alanine, serine, taurine, phenylalanine, tyrosine, proline, histidine, glycine, and valine, against a background of 90.4±2.1 mmol/L ammonia are reduced critically.
 Conclusion: Zoledronic acid initiates an aseptic chronic inflammatory process in the jaw bones, accompanied by toxic acidosis, which worsens osteoporotic symptoms.

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis.

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.

Chiral Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis (CE) is a well-established and one of the most powerful separation techniques in the field of chiral separations. Its hyphenation with mass spectrometry (MS) combines both the high separation efficiency and low sample consumption of CE and the high sensitivity and structural information of MS. Thus, the outstanding chiral resolution power of CE along with the MS advantages makes CE-MS a perfect combination to achieve sensitive enantioseparations. This chapter describes three representative examples of different approaches used in the chiral analysis of amino acids in biological fluids by CE-MS. The first methodology uses the partial filling technique to avoid the entry of cyclodextrins in the MS source. The second method shows the possibility to carry out the direct coupling EKC-MS even when a relative high concentration of a native cyclodextrin is used as chiral selector. The last example illustrates an alternative strategy based on the formation of stable diastereomers between an enantiomerically pure chiral reagent and the amino acids enantiomers which can be separated in an achiral environment.

Direct Detection of Amino Acids Using Extractive Electrospray Ionization Tandem Mass Spectrometry

Abstract A novel method was developed for the direct detection of amino acids in biological fluids by extractive electrospray ionization (EESI) tandem mass spectrometry (MS) with minimal sample pretreatment. Based on our previous research, a novel EESI ion source was constructed to improve analytical performance and operation safety. The EESI-MS conditions were optimized using representative amino acid standards. The methanol-water reagent solvent (1:1, v/v) was electrosprayed at 5 μL min−1 at a high voltage (+4 kV, positive ion detection mode). The temperature of the heated capillary was optimized to be 150 °C. Collision induced dissociation (CID) experiments were performed by applying 17%−25% of the collision energy to the precursor ions isolated with a window width of 1.5 mass/charge (m/z) units. The limit of detection (LOD) for these amino acids was in the range of 0.14−26.2 μg L−1, and the linear dynamic range was larger than two orders of magnitude. The average time for a single amino acid analysis using a typical human urine sample was less than 0.5 min. The recovery rates for different concentrations of amino acids ranged from 82.6% to 105.6%, and the relative standard deviations (RSD) ranged from 2.2% to 11.4%. The results showed that EESI-MS is a powerful tool for the rapid, sensitive, and quantitative detection of amino acids in complex biological samples.

Automated GC–MS analysis of free amino acids in biological fluids

A gas chromatography–mass spectrometry (GC–MS) method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate is carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing automation of the entire procedure, including addition of reagents, extraction and injection into the GC–MS. The total analysis time was 30 min and 30 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03–12 μM and 0.3–30 μM, respectively. The method was validated using a certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intra-day precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0–8.8%, 0.9–8.3%, and 2.0–14.3%, respectively, while the inter-day precision for human urine was 1.5–14.1%.

Analysis of 25 underivatized amino acids in human plasma using ion‐pairing reversed‐phase liquid chromatography/time‐of‐flight mass spectrometry

Amino acids in biological fluids have previously been shown to be detectable using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with perfluorinated acids as ion-pairing agents. To date, these studies have used precursor mass, retention time and tandem mass spectrometry (MS/MS) to identify and quantify amino acids. While this is a potentially powerful technique, we sought to adapt the method to time-of-flight (TOF)MS. A new application of a recently described liquid chromatographic separation method was coupled with TOFMS to employ accurate mass for qualitative identification; resulting in additional qualitative data not available with standard single quadrupole data. In the current study, we evaluated 25 physiological amino acids and one dipeptide that are routinely quantified in human plasma. Accuracy and precision of the method was evaluated by spiking human plasma with a mix of the 25 amino acids; in addition, the inclusion of a cation-exchange cleanup step was evaluated. The calibration curves were linear over a range from 1.56 to 400 microM. The dynamic range was found to be within physiological levels for all amino acids analyzed. Accuracy and precision for most of the amino acids was between 80-120% spike recovery and <10% relative standard deviation (RSD). The LC/MS technique described in this study relies on mass accuracy and is suitable for the quantitation of free amino acids in plasma.

Determination of excitatory amino acids in biological fluids by capillary electrophoresis with optical fiber light-emitting diode induced fluorescence detection

The capillary electrophoresis (CE) system with optical fiber light-emitting diode (optical fiber LED) induced fluorescence detector was developed for the analysis of the excitatory amino acids (EAAs) tagged with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of EAAs was carried out in an uncoated fused-silica capillary (50 cm x 75 microm i.d.) with a buffer of 10 mM borate at pH 9.3 and an applied voltage of 20 kV. High sensitivity was obtained by the use of optical fiber LED induced fluorescence detector with a violet LED as the excitation light source. The limits of detection (S/N = 3) for glutamic acid (Glu) and aspartic acid (Asp) were 2.1 x 10(-8) and 2.3 x 10(-8) M, respectively. The detection approach was successfully applied to the analysis of Glu and Asp in biological fluids including human serum, rabbit serum and human cerebrospinal fluid (CSF) with satisfactory results.

A new reversed‐phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism

Seventy-six compounds of biological interest for the diagnosis of inherited disorders of amino acids (AA) metabolism have previously been demonstrated to be detectable in positive mode electrospray ionisation tandem mass spectrometry (ESI-MS/MS), after separation by ion-pairing reversed-phase liquid chromatography (RPLC). The separation method used tridecafluoroheptanoic acid as ion-pairing agent, and a gradient of acetonitrile for the elution of the most retained compounds. This method had previously been demonstrated to be suitable for the qualitative diagnosis of many AA disorders, and for the quantitative measurement of 16 AA in biological fluids, using their stable isotope labelled (SIL) AA as internal standard. For quantification of the other AA, an internal standard was chosen among the available SIL-AA, as close as possible to the analyte to be measured, in terms of structural analogy, and of retention time in the chromatographic system. The performances of the quantitative analysis of the other AA to be measured are reported here. They show validated results for several AA, allowing their accurate quantification, with another SIL-AA as internal standard. For some other AA, quantitative results were not accurate, allowing only semi-quantitative or qualitative determination for these parameters.

Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids d-alanine, d-glutamine, and d-aspartic acid have been observed in cerebrospinal fluid , and d-alanine and d-glutamic acid in urine . To the best of our knowledge no measurements of either d-alanine in cerebrospinal fluid or d-glutamic acid in urine have been presented in the literature before.

Encefalomiopatía mitocondrial, acidosis láctica y accidentes cerebrovasculares (MELAS) en edad pediátrica con la mutación A3243G en el gen del ARNtLeu(UUR) del ADN mitocondrial

To evaluate three patients with the mitochondrial encephalopathy, lactic acidosis and cerebrovascular accident syndrome (MELAS) diagnosed in childhood, with particular reference to the initial symptoms and clinical evolution during the first stage at a pediatric age, and to compare them with other studies on the subject. Two boys and a girl of 10, 11 and 13 years had tests on lactate, pyruvate and aminoacids in biological fluids under basal conditions and also functional tests and enzyme activity assay of the mitochondrial respiratory chain of a muscle biopsy. We also analysed the particular DNA mutations related to MELAS in different tissues from these patients and in lymphocytes from members of the mothers' families who could be tested. The patients fulfilled the clinical criteria for the MELAS syndrome. Neuroimaging showed cerebrovascular accidents. Neurophysiological studies showed myopathy in one patient and neuroaxonal neuropathy in another. In two cases ophthalmological study showed retinitis pigmentaria and during cerebrovascular accidents transient phenomena of homonymous hemianopsia and cortical blindness were seen. In all patients muscle biopsy showed ragged red fibres and the biochemical study showed an enzyme deficit in the respiratory mitochondrial chain. On molecular genetic study of the mitochondrial DNA (mtDNA) there was a particular mutation A3243G on the tRNA(Leu) in all patients and some members of the mothers' families. In children with frequent episodes of migraine headaches, vomiting, refractory epilepsy and fatigue the presence of a mitochondrial disease should be suspected. On detection of mtDNA mutations MELAS may be diagnosed even without all the clinical criteria which characterise this syndrome.

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N( O, S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6–150 pg injected. The calibration curves for each amino acid in the range of 0.02–2 μg were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83–112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3–8.9% ( n = 3) and 1.9–15.8% ( n = 3) respectively.

Analysis of Amino Acids in Biological Fluids by Pentafluorobenzyl Chloroformate Derivatization and Detection by Electron Capture Negative Ionization Mass Spectrometry

Pentafluorobenzyl chloroformate (PFBCF) has been utilized as a derivatization reagent for amino acids (AAs) in biological fluids with subsequent detection by electron capture negative ionization mass spectrometry (ECNI/MS). AAs were derivatized in one step in aqueous solution, plasma, and whole blood at room temperature. To demonstrate quantitative analysis, phenylalanine concentrations were determined in human plasma. AAs were derivatized in one step using PFBCF and a mixture of water, ethanol, and pyridine/dimethylaminopyridine. TheN-pentafluorobenzyloxycarbonyl amino acid ethyl esters (fφ-AA-OEt) exhibited good GC properties and the ECNI mass spectra are dominated by the [M-181]−ion. Thefφ-AA-OEt derivatives can be easily detected at the femtomole level by selected ion monitoring. Phenethyl alcohol was also derivatized, using anhydrous conditions, and the resulting PFB carbonate's ECNI mass spectrum was dominated by the [M-181]−ion. The ECNI molar response of the PFB carbonate derivative is two times that of the corresponding pentafluorobenzoate.

Quantitative Methods for Amino Acid Analysis in Biological Fluids

We must be able to provide accurate quantitative analyses of free amino acids in biological fluids. These are needed to: diagnose inherited metabolic disorders and to control their treatment; to monitor patients requiring long-term nutritional support; to evaluate new baby milks and parenteral or formulated enteral feeds; to detect renal drug toxicity; and to monitor bone or muscle turnover. Sometimes it is enough to analyse only one or a small group of amino acids, but more often, a full amino acid profile is required. This is an extremely difficult task as there are some 40 or 50 amino acids or amino acid conjugates in biological fluids, I with more than a WOO-fold range of concentrations. In order to separate amino acids for analyses, we have to exploit very small differences in structure and ionisation. Success depends upon fastidious attention to detail, not only in the analyses, but also in the collection and preparation of samples.? The results can only be interpreted against a sound knowledge of metabolism. Many clinicians lean heavily upon the laboratories for interpretation, and it is essential that the data are presented in a way that is helpful. Quantitative analyses for clinical use became feasible 40 years ago when Moore and Stein reported the separation of standard and plasma amino acids with a sulfonated polystyrene resin column.v' Spackman et al? automated this procedure. Among clinical laboratories, cationexchange is still by far the commonest method

Benzoylation of sugars, polyols and amino acids in biological fluids for high-performance liquid chromatographic analysis

A precolumn benzoylation for analyzing sugars, polyols and neutral amino acids in biological fluids by high-performance liquid chromatography has been developed, which avoids protein precipitation, drying procedures and the use of pyridine. Derivatization and chromatography can be performed within one hour with a minimum detectable quantity of ca. 1 pmol (signal-to-noise ratio > 2). The derivatization products of glucose, mannitol and neutral amino acids were identified by electrospray mass spectrometry (ES-MS) to be tetra- and pentabenzoyl glucose, penta- and hexabenzoyl mannitol and 2-phenyl-5-benzoyloxyoxazoles, respectively.

Intra- and interlaboratory quality control for assay of amino acids in biological fluids: 14 years of the French experience

The functioning of an external quality-control scheme for amino acids set up in 1978 is described. Two measurements were made each month by participating laboratories on a control plasma sample provided by the quality-control center; freeze-dried samples were used from 1978 to 1989 and liquid samples since 1990. In addition, two "blind" samples were sent to the laboratories each year. Every 3 months, the overall results and those of the individual laboratories were analyzed statistically. The validity of the liquid sample control is demonstrated. The progressive improvement of results is commendable. In 1990, the coefficients of variation for all participants ranged from 8.4% for alanine to 23.5% for methionine. The standards used for calibration could contribute to the broad range of results, especially those for histidine and ornithine. The use of blind samples made it possible to detect problems of calibration, of linearity of measurement, of contamination, and of identification of unusual amino acids.

Quantitation of tryptophan and other plasma amino acids by automated pre-column o-phthaldialdehyde derivatization high-performance liquid chromatography: improved sample preparation

Pre-column derivatization with o-phthaldialdehyde is a rapid and sensitive method for the quantitation of amino acids in biological fluids. This method uses acetonitrile as a deproteinizing reagent which gives improved recovery of tryptophan compared with 5-sulfosalicylic acid and permits the measurement of aspartic acid which coelutes with 5-sulfosalicylic acid. The method is automated to increase reproducibility and convenience. Mean coefficients of variation for peak areas relative to internal standard were 3.2 and 5.2% for amino acid standards and plasma samples, respectively. The presence of nitrilotriacetic acid stabilized the o-phthaldialdehyde reagent which is important in an automated system. The method is suitable for the analysis of large numbers of plasma samples where total tryptophan and aspartic acid are of interest.

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH 2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.

An ultra rapid and sensitive high-performance liquid chromatographic method for determination of tissue and plasma free amino acids

An ultra rapid and sensitive HPLC method for measuring individual free amino acids in biological fluids has been developed by using o-phthaldialdehyde/3-mercaptopropionic acid as derivatization agent and employing 3-μm-particle-size reversed-phase columns. Resolution of the amino acid derivatives is accomplished with an acetonitrile gradient in 12.5 m m sodium phosphate buffer, pH 7.2. These conditions facilitate separation of the 23 major tissue free physiological amino acids in the lower picomole range in less than 13 min. Muscle, liver, and kidney free amino acid concentrations, as determined by HPLC, are in the expected physiological range and compare favorably with those obtained by conventional amino acid analyzer.

Automated Analysis of O-Phthalal-Dehyde Derivatives of Amino Acids in Physiological Fluids by Reverse Phase High Performance Liquid Chromatography

Abstract An automated method is described for the determination of free amino acids in biological fluids using precolumn deriva-tization with o-phthalaldehyde and reverse phase high performance liquid chromatography. Chromatographic separation of amino acids is accomplished in 24 minutes (cycle time 44 minutes). As little as 1.5 pmol of most commonly occurring amino acids can be accurately quantified. Accuracy and reproducibility are optimized by automating the derivatization-injection sequence and by correcting for variations in the fluorescence response of each amino acid in each run. A total of 31 analyses can be completed in 24 hours on a single column (7 standards and 24 unknowns). The method can be used in the general determination of free amino acids in biological fluids, or can be further accelerated and used for the quantitation of specific amino acids simply by altering the elution conditions.

High performance, ion-exchange chromatography of amino-acids in biological fluids using Chromakon 500- performance of the apparatus

High performance, ion-exchange chromatography of amino-acids in biological fluids using Chromakon 500- performance of the apparatus